Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Heterelogous Expression of Plant Genes

Heterelogous Expression of Plant Genes Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2009, Article ID 296482, 16 pages doi:10.1155/2009/296482 Review Article Filiz Yesilirmak and Zehra Sayers Faculty of Engineering and Natural Sciences, Sabanci University, Orhanli, Tuzla, 34956 Istanbul, Turkey Correspondence should be addressed to Filiz Yesilirmak, filizy@sabanciuniv.edu Received 30 January 2009; Accepted 24 May 2009 Recommended by Pushpendra Gupta Heterologous expression allows the production of plant proteins in an organism which is simpler than the natural source. This technology is widely used for large-scale purification of plant proteins from microorganisms for biochemical and biophysical analyses. Additionally expression in well-defined model organisms provides insights into the functions of proteins in complex pathways. The present review gives an overview of recombinant plant protein production methods using bacteria, yeast, insect cells, and Xenopus laevis oocytes and discusses the advantages of each system for functional studies and protein characterization. Copyright © 2009 F. Yesilirmak and Z. Sayers. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction posttranslational modifications, efficiency of the expression system, as well as simplicity and cost are discussed in the following sections. Heterologous expression involves identification of genes and transfer of the corresponding DNA fragments to hosts other than the original source for synthesis of the encoded 2. Principal Components of proteins. Protein isolation, especially from plant sources, Heterologous Expression can be costly, cumbersome and lengthy, and heterologous expression provides a convenient alternative. This method- Basic principles of heterologous cloning and expression ology allows large-scale production of plant proteins in are summarized in Figure 1. Major parameters that affect microorganisms to study their biochemical and biophysical choices at different stages are also indicated. The choice of features. Foreign hosts may also provide a simpler system for the expression system and vector is a critical step in this studies on functions of proteins and for elucidation of their procedure and, as indicated, advantages and disadvantages roles in complex mechanisms such as metabolic reactions of several factors have to be considered. Expression systems and membrane transport. Recombinant plant proteins and are selected depending on whether the purpose of study is peptides produced by heterologous expression are also used production of large quantities of protein or investigation of in industrial applications. Examples are provided by the functional features of the cloned protein. The physicochem- synthesis of a medicinal peptide from ginseng as potential ical properties of the investigated protein also play a role in drug against diabetes [1] or production of plant lectins [2]in this choice. A general review of frequently used expression both cases in yeast. systems is provided by Yin et al. [3]. The present review covers the recent literature on A comprehensive survey of commercially available plant gene expression in bacteria, yeast, insect cells and expression vectors has recently been published [4]. The Xenopus oocytes and presents the comparative advantages most commonly used vectors are fusion systems that link and disadvantages of each system. It also provides a survey additional amino acid sequences (tags) to the protein of recent examples of application of heterologous expression through a recognition site for a specific protease. Tags may technology to plant proteins. A comprehensive list of plant consist of a short peptide sequence or a full protein which proteins expressed heterologously is given in Table 1.Factors can be cleaved from the protein when desired. Presence influencing the choice of hosts, including the stability of tag sequences facilitates solubility, purification, quan- and folding characteristics of the protein, requirement for tification, identification, localization, and assaying of the 2 International Journal of Plant Genomics Gene(s) •Protein modifications • Solubility • Yield • Fusion partners Host Vector • Cost • Stable expression • Efficiency •Transient expression • Environment/safety/ health Host Vector Expression No Expressed Functional studies Yes (in vivo) Purification •Protein analysis •Structural analysis •Production • Functional studies (in vitro) Figure 1: Flow chart for heterologous expression. expressed protein. Frequently used fusion partners include and relatively low cost. Almost all commercially available glutathione-S-transferease (GST), his-tag (poly-histidines), inducible cloning vectors are compatible with E. coli and maltose binding protein (MBP), thioredoxin (TrxA), FLAG extensive biochemical and genetic information is available. epitope-tag, c-Myc epitope-tag, disulfide isomerase I (DsbA), One of the disadvantages of using E. coli as an expression polyarginine-tag (Arg-tag), calmodulin-binding peptide, host arises from its inability to perform post-translational cellulose-binding domain, poly-histidine affinity tag (HAT- modifications, which are often required for correct fold- tag), N-utilizing substance-A (NusA), S-tag, streptavidin- ing and functional activity of the recombinant protein. binding peptide (SBP-tag), strep-tag, fluorescent proteins This applies particularly to some membrane proteins and (e.g., green fluorescent protein (GFP)) and ubiquitin [4]. enzymes [3]. Another disadvantage is that E. coli is generally MBP and NusA are specifically used to increase the solubility. not suitable for proteins which contain many disulfide MBP is considered to be much more effective for enhancing bonds or require glycosylation, proline cis/trans isomer- solubility than GST and thioredoxin [5]. The major dis- ization, disulfide isomerization, lipidation, sulphation, or advantages of fusion protein systems are the requirement phosphorylation [8]. Some eukaryotic proteins that retain of expensive proteases for cleavage from the recombinant their full biological activity in the nonglycosylated form protein and the low yield of cleavage reactions [6]. have,however,beenproducedin E. coli. The unglycosylated Depending on the host system, vectors for transient or human growth hormone (hGH) binding protein secreted stable expression can be chosen as indicated below. from E.coli retains the same binding affinity and specificity as the wild-type hGH binding protein suggesting that recombinant protein is properly folded and glycosylation is 3. Expression Hosts not required for binding [9]. Production of proteins that are stabilized by disulfide 3.1. Prokaryotic Expression Systems bonds in E.coli often results in proteolytic degradation or 3.1.1. Escherichia Coli. Escherichia coli (E. coli) is the first misfolding and formation of inclusion bodies [6]. One and most extensively used prokaryotic expression system for strategy developed to improve this situation is to target these proteins to the periplasm where the nonreducing heterologous protein production [7]. It remains generally the first choice due to its simplicity, rapid growth rate, environment allows formation of disulfide bonds [10, 11]. In International Journal of Plant Genomics 3 addition, the E.coli periplasm contains chaperone-like disulfide-binding proteins (DsbA, DsbB, DsbC, and DsbD), folding catalysts, and peptidyl-prolyl isomerases (SurA, Cd/protein RotA, FklB, and FkpA) that support disulfide bond forma- GSTdMT 3.4 ± 1 tion and are important for correct folding of periplasmic proteins [12–14]. Disulfide bond formation is achieved via fusion to DsbA or DsbC [15, 16] and periplasmic secretion results in the functional production of a variety of recombinant proteins [17]. In a recent study, the rescue of unstable lipase B from Pseudozyma antarctica (PalB), with periplasmic folding factors was demonstrated [18]. 2 Another strategy involves the use of the trxB gor dou- ble mutant lacking thioredoxin reductase and glutathione reductase genes [19, 20]. This double mutant was used for 220 240 260 280 300 heterologous expression of barley oxalate oxidase (HvOXO) Wavelength (nm) in E.coli [21]. Thegenefor an osmotin-like cryoprotective Figure 2: UV-visible absorption spectrum of GSTdMT at 2.7 protein from Solanum dulcamara was expressed in E.coli mg/mL concentration in 20 mM HEPES buffer at pH 8.0. The and directed to periplasmic localization using an expression charge transfer band between 240 and 260 nm due to Cd-S vector containing the pelB signal sequence [22]. This resulted interaction is indicated by the arrow. The Cd/protein ratio is given in high concentrations of soluble protein with cryoprotective in the inset. activity, whereas expression in the bacterial cytoplasm only yielded large amounts of insoluble and aggregated protein. The seagrass HAK K transporters, CnHAK1 and CnHAK2 Some of the plant proteins accumulated in insoluble were also overexpressed in E.coli and it was found that inclusion bodies in E.coli can be solubilized and refolded CnHAK1, but not CnHAK2, mediated very rapid K or Rb to restore activity after purification from the host. Exam- influxes [35]. Using a dicarboxylate uptake-deficient E.coli ples include Arabidopsis thaumatin-like protein (ATLP3) mutant, a peptide transporter, AgDCAT1 from alder, was which was purified from inclusion bodies and the refolded shown to be a dicarboxylate, including malate, succinate, form displayed activity against some pathogenic fungi [23]. fumarate, and oxaloacetate, transporter [36]. To validate the potential antifungal activity of Solanum E.coli has also been used for expression of small plant nigrum osmotin-like protein (SnOLP) was overexpressed proteins with a fusion partner. Metallothioneins (MTs), in E.coli and the recombinant protein was refolded using which are difficult to purify from natural sources because reduced:oxidized gluthatione redox buffer and its in vitro of their small molecular weight (7 kD), unusual amino acid activity was demonstrated [24]. The soybean RHG1-LRR sequences containing a large number of cysteins and their domain protein was solubilized from inclusion bodies using proteolytic susceptibility belong to this class. Several MTs urea and refolded by removing the urea in the presence of 2+ including a Cd binding Type 1 durum wheat metalloth- arginine and reduced/oxidized glutathione [25]. ionein (dMT) [37], fava bean Type 1 and Type 2 MTs [38], Many plant enzymes are expressed in insoluble inclusion Arabidopsis MT1, MT2 and MT3 proteins [39], Type 3 MT3- bodies but it is still possible to obtain high yields of active A from the oil palm [40], Type 2 MT, QsMT from Quercus forms for structural studies [26]. Thematurepolypeptide of suber [41]havebeenproducedin E.coli mainly for structural FatB thioesterase from the developing seed tissues of Mad- analyses. Since the fusion constructs of durum MT with huca butyracea was characterized by heterologous expression GST (GSTdMT) can be purified in well-defined oligomeric in E.coli [27]. The functionality of the MbFatB in the heterol- states they are used as model systems for studies on metal- ogous system was revealed by the altered growth behavior binding and for structural analyses. Figure 2 illustrates that and cell morphology of the bacteria due to the changes in Cd-binding to GSTdMT can be detected by UV-visible the fatty acid profile. The maize chloroplast transglutaminase spectroscopy. The metal content of GSTdMT was shown to (TGZ) [26] and glutamatecysteine ligase (GCL) [28]were be the same as that expected from dMT alone. An example of efficiently overexpressed in E.coli. Recently, DELLA proteins the shape models generated from X-ray solution scattering from both Arabidopsis and Malus domestica, which are data for GSTdMT is shown in Figure 3, together with the involved in regulation of plant growth in response to phyto- fit to experimental data. The models support a fold for hormonal signals, were isolated and expressed in E. coli [29]. dMT similar to that expected for the free molecule [42, 43]. Examples of functional expression of plant proteins in These results are in agreement with earlier work suggesting E.coli are provided mostly by studies on membrane proteins. independent folding of GST and its fusion components [44] Amutantwithverylow K uptake was used as host and indicate that recombinant fusion complexes are useful as for studies on the K transporters AKT2 [30], AtKUP1- model systems for structural studies. 2[31], AtHKT1 [32] from Arabidopsis and EcHKT1 and EcHKT2 from Eucalyptus camaldulensis [33]. In another example, E.coli C43 strain, which is suitable for expression of 3.2. Eukaryotic Expression Systems. Eukaryotic expression membrane proteins was used for functional characterization systems offer the possibility of posttranslational modifi- of chloroplast ATP/ADP transporter from Arabidopsis [34]. cations and are often used for investigations of protein Absorbance (a.u.) 4 International Journal of Plant Genomics Table 1: Heterologous expression of plant proteins grouped according to the host cells. Protein expressed Plant Reference Escherichia coli Lipase B (PalB) Pseudozyma antarctica [18] Hordeum vulgare, Triticum Oxalate oxidase [21] aestivum Osmotin-like Solanum dulcamara [22] cryoprotective protein Thaumatin-like protein Arabidopsis thaliana [23] (ATLP3) Osmotin-like protein Solanum nigrum [24] (SnOLP) RHG1-LRR domain Glycine max [25] Chloroplast Zea mays [26] transglutaminase (TGZ) FatB thioesterase Madhuca butyracea [27] Glutamatecysteine ligase Arabidopsis thaliana [28] (GCL) Arabidopsis thaliana , Malus DELLA proteins [29] domestica K transporters; KAT1, AKT2-3, AtKUP1/AtKT1/AtPOT1, Arabidopsis thaliana [30–32] AtKUP2/AtKT2/AtPOT2, AtHKT1 K transporters, EcHKT1, Eucalyptus globulus [33] EcHKT2 ATP/ADP transporter Arabidopsis thaliana [34] HAK K transporters, Cymodocea nodosa [35] CnHAK1,CnHAK2 Peptide transporter family Alnus glutinosa [36] member, AgDCAT1 Type 1 MT, dMT Triticum durum [37] Type 1 and Type 2 MTs Vicia faba [38] MT1, MT2, and MT3 Arabidopsis thaliana [39] Type 3 MT3-A Elaeis guineensis [40] Type 2 MT, QsMT Quercus suber [41] Soybean seed ferritin Glycine max [126] Saccharomyces cerevisiae H -amino acid symporter Arabidopsis thaliana [47] and K channel, KATl Phosphate transporters; Arabidopsis thaliana [48] AtPT1 and AtPT2 K transporter, HvHAKI Hordeum vulgare [49] K transporters, AtKT1, Arabidopsis thaliana [50, 51] and AtKT2, AtKUP1 K transporter, HKT1 Triticum aestivum [52, 53] Sulfate transporters, Lycopersicon esculentum [54] LeST1-1 and LeST1-2 Copper transporters, Arabidopsis thaliana [55] (COPT1–5) Peptide transporter, Arabidopsis thaliana [56] AtPTR1 + + K /H antiporter, AtChx17 Arabidopsis thaliana [57] International Journal of Plant Genomics 5 Table 1: Continued. Protein expressed Plant Reference Hexose transporters, Vitis vinifera [58] VvHT4 and VvHT5 Plasma membrane-localized Arabidopsis thaliana [59] H /inositol symporter, AtINT2 High affinity Arabidopsis thaliana [60] GABAtransporter, AtGAT1 Tonoplast Intrinsic Proteins, AtTIP2;1 and Arabidopsis thaliana [61] AtTIP2;3 Sorbitol transporters, Plantago major [62] PmPLT1 and PmPLT2 Pichia pastoris Nitrate reductase Spinacia oleracea,Zea mays [69] Invertase Ipomoea batatas [70] α1,6-galactosyltransferase Trigonella foenum-graecum [71] α1,6-xylosyltransferase Arabidopsis thaliana [72] Arabidopsis thaliana Bos taurus, Drosophila Glycosyltransferases melanogaster, [73] Caenorhabditis elegans, Leucopersicon esculentum β-D-fructofuranosidase Oryza sativa [74] Apyrase Solanum tuberosum [75] Oxalate oxidases, HvOXO, Hordeum vulgare, Triticum [76, 77] TaOXO aestivum Canavalia brasiliensis, Lectin [2, 79] Nicotiana tabacum Low-affinity cation Triticum aestivum [80] transporter (LCT1) 2S albumin storage Glycine max [127] proteins (AL1 and AL3) Baculovirus-mediated insect cell Patatin Solanum tuberosum [81] Reductase isoforms, AR1 Arabidopsis thaliana [82] and AR2 Peroxisomal short-chain Arabidopsis thaliana [83] acyl-CoA oxidase A Cyclin-dependent kinase A Arabidopsis thaliana [84] (CDKA) NADH-cytochrome (Cyt) Arabidopsis thaliana [85] b5 reductase Geranylgeranyltransferase-I Arabidopsis thaliana [86] (GGT-I) Acyl-CoA synthetase Arabidopsis thaliana [87] Homogentisate Arabidopsis thaliana [88] phytyltransferase (+)-Abscisic Acid Arabidopsis thaliana [89] 8’-Hydroxylase β1,2-xylosyltransferase Arabidopsis thaliana [90] Ethylene-inducing xylanase Nicotiana tabacum [91] 6 International Journal of Plant Genomics Table 1: Continued. Protein expressed Plant Reference ADP-glucose pyrophoshorylase Hordeum vulgare [92] (AGPase) K channels, AKT1, KAT1, Arabidopsis thaliana [93–95] and KCO1 K channels KST1, SKT1, Solanum tuberosum [96, 97] and KST1 Transporter AUX1 Arabidopsis thaliana [98] β-phaseolin polypeptides Phaseolus vulgaris [128] Ac-specific ORFa protein, Zea mays [129] Cysteine protease papain Carica papaya [130] Mitochondrial protein Zea mays [131] URF13 LAT52 protein Lycopersicon esculentum [132] Auxin-binding protein Zea mays, Nicotiana [133, 134] (ABP1) tabacum Calreticulin and auxin Zea mays [135] binding protein Cinnamate 4-Hydroxylase Arabidopsis thaliana [136] Cryptochrome-1 Arabidopsis thaliana [137] Phototropin 2 Arabidopsis thaliana [138] Histidinol dehydrogenase Brassica oleracea [139] Putative soluble epoxide Solanum tuberosum [140] hydrolase (sEH) lmidazoleglycerolphosphate Arabidopsis thaliana [141] dehydratase Phytone synthase, Narcissus pseudonarcissus [142, 143] Phytoene desaturase 4-coumarate:coenzyme A Populus trichocarpa, [144] ligase (4Cl) Populusdeltoides Xenopuslaevisoocytes Na − K cotransporter Arabidopsis thaliana [39] HKT1 AgDCAT1 nodule-specific Alnus glutinosa [43] transporter AtNAR2.1/AtNRT2 Nitrate Arabidopsis thaliana [102] Transport System HKT Constructs, Triticum aestivum, [103] AtHKT1 HKT1 chimeras Arabidopsis thaliana HKT1 superfamily of Eucalyptus camaldulensis [104] K /Na transporters Ammonium transporter, Lycopersicon esculentum [105] LeAMT1 Ammonium transporter, Arabidopsis thaliana [106] AtAMT1;2 Sucrose transporters, Arabidopsis thaliana,Lotus [107–109] AtSUC2, AtSUC9, LjSUT4 japonicus Al-activated malate transporter, Brassica napus, Triticum [110, 111] BnALMT1,BnALMT2, aestivum ALMT1 Polyol transporters, Arabidopsis thaliana, [112, 113] AtPLT5, PmPLT1 Plantago major International Journal of Plant Genomics 7 Table 1: Continued. Protein expressed Plant Reference Inositol transporter2, Arabidopsis thaliana [114, 115] AtINT2, AtINT4 Amino acid transporter, Arabidopsis thaliana [116] AtCAT6, Cation–Cl- cotransporter, Arabidopsis thaliana [117] CCC Anion-selective Zea mays [118] transporter, ZmALMT1 K channel, SIRK Vitis vinifera [145] K channel, KZM1 Zea mays [146] K channel, ZMK1 Zea mays [147] K channels, SKT1 and Solanum tuberesum, [148] LKT1 Lycopersicon esculentum AKT2-KAT2 subunitits Arabidopsis thaliana [149] K channel, KAT1 Arabidopsis thaliana [150] Cyclic nucleotide-gated ion Arabidopsis thaliana, channels AtCNGC2, [151, 152] Nicotiana tobacum AtCNGC1, -2 Putative transporter Glycine max [153] (GmN70) Al-activated malate Triticum aestivum [154] transporter, TaALMT1 High affinity γ-aminobutyric acid Arabidopsis thaliana [155] transporter, AtGAT1 Aquaporins, ZmPIP1a, ZmPIP1b, ZmPIP2a, PIP1, Zea mays [124, 156, 157] ZmPIP2;1 Aquaporin, PIP1 Lycopersicon esculentum [158] Aquaporin, PIP2 Juglans regia [159] Tonoplast intrinsic protein, Arabidopsis thaliana [160, 161] AtTIP2;1 Mesembryanthemum Aquaporin, McTIP1;2 [162] crystallinum Aquaporin, HvPIP1;6 Hordeum vulgare [163] Tonoplast intrinsic protein, Panax ginseng [164] PgTIP1 Nodulin 26 intrinsic Arabidopsis thaliana [165] protein, AtNIP2;1 PIP-1-type; NtPIP1;1, NtAQP1; PIP-2-type; Nicotiana tabacum [166] NtPIP2;1 CjMDR1, ATP-binding Coptis japonica [167] cassette protein GlpF-like intrinsic protein Physcomitrella patens [168] (GIP1;1), Metal tolerance protein1, Arabidopsis thaliana [169] AtMTP1 AtTPK4 tandem-pore Arabidopsis thaliana [170] K channel FRD3, multidrug and toxin Arabidopsis thaliana [171] efflux (MATE) 8 International Journal of Plant Genomics −1 −2 −3 0 0.1 0.2 0.3 −1 S (nm ) (a) (b) Figure 3: A: a low resolution shape model for GSTdMT. The GST dimer (red and blue) is located at the center from which the dMT molecules extend (green). B: the scattering curve expected from the model (−) agrees well with the experimental data (...). I(S) is the scattering intensity and S the scattering vector given by S = 4π sinθ/λ, where 2θ is the scattering angle and λ= 1.5 A is the wavelength of X-rays. The model and the expected scattering pattern were calculated using the programs in the ATSAS package (EMBL Hamburg Outstation). function. Processing reactions such as O-and N -linked gly- bidopsis [51] also utilized S. cerevisiae mutants. Another K cosylation, tyrosine, serine, and threonine phosphorylation, transporter characterized in this system is HKT1 from wheat addition of fatty acid chains, processing of signal sequences, [52, 53]. Kinetic uptake analyses of tomato sulfate trans- disulfide bond formation, and correct folding can all be porters, LeST1-1 and LeST1-2 were carried out using the S. readily performed in eukaryotic hosts. The most commonly cerevisiae sulfate transporter mutant [54]. The five members used eukaryotic systems are yeast, insect, mammalian, and of the copper transporter family COPT1–5 from Arabidopsis plant cells. were characterized using a copper transport null mutant [55]. A peptide transporter AtPTR1 gene from Arabidopsis 3.2.1. Yeast. As a single cell eukaryotic organism, yeast has was isolated and complemented in a peptide transport- + + deficient mutant [56]. A putative K /H antiporter, AtChx17 molecular, genetic, and biochemical characteristics which are similar to those of higher eukaryotes, and is useful for was heterologously expressed and characterized in an S. heterelogous protein production. Yeast cells can grow rapidly cerevisiae kha1 deletion mutant [57]. To test their functional activity, the grapevine hexose transporters VvHT3, VvHT4, with high cell densities, and are easy to manipulate and yeast cultures are cost effective. The two most commonly used and VvHT5 were expressed in the S. cerevisiae mutant EBY.VW4000, which is deficient in glucose transport due organisms are Saccharomyces cerevisiae (S. cerevisiae)and Pichia pastoris (P. pastoris) [7]. to concurrent knock-out of 20 endogenous transporter genes [58]. Growth-based complementation assays were used to demonstrate function of the transporters but resulted Saccharomyces Cerevisiae. Baker’s yeast, S. cerevisiae,is widely used as a host organism for heterologous expression in inadequate rates of glucose uptake. A more sensitive assay based on direct measurement of radioactively labelled of proteins. Its genetics and physiology are well documented glucose uptake revealed that this mutant expressing VvHT4 and proteins are posttranslationally modified through the and VvHT5 accumulated labelled glucose at higher rates than mechanisms similar to those found in plants. The limitations yeast transformed with the empty vector, demonstrating of this host system are low yields, cell stress due to the pres- the functionality of the glucose transporters. Although ence of the foreign gene and hyperglycosylation of secreted VvHT3:GFP (green fluorescent protein) fusion protein was foreign proteins. Lack of a strong inducible promoter can be targeted to the plasma membrane in plant cells, VvHT3 was circumvented using P. pastoris [45]. Earlier work on heterelogous expression for screening of found not to be functional in the yeast system [58].Yeast expression studies were, in several instances, complemented plant cDNA libraries by complementation in S. cerevisiae by studies in other organisms to verify functional and kinetic null mutants was reviewed by Frommer and Ninnemann [7]. The S. cerevisiae mutants provide a convenient system properties of recombinant proteins. The plasma membrane- localized H /inositol symporter AtINT2 of Arabidopsis was for functional and kinetic studies of transporters [46]. The studied by expression in an inositol uptake/inositol biosyn- electrophysiological properties of membrane transporters, + + thesis double mutant in S. cerevisiae and in Xenopus oocytes H -amino acid symporter and K channel, KAT1 [47]and [59]. In this study, the amount of AtINT2 protein in yeast phosphate transporters; AtPT1 and AtPT2 of Arabidopsis were characterized using S. cerevisiae [48]. Recently, func- plasma membrane was sufficient for complementation, but not for functional and kinetic analyses. In oocytes, however, tional expression of transporters such as an HvHAKI from it was possible to show that AtINT2 mediated the symport barley [49], AtKT1 and AtKT2 [50], and AtKUP1 from Ara- log I (S) International Journal of Plant Genomics 9 of H [59]. Expression and functional characterization of [73]. To confirm that Osβfruct3 from rice encoded a vac- Arabidopsis AtGAT1 in S. cerevisiae and Xenopus oocytes uolar type β-D-fructofuranosidase, the Osβfruct3 cDNA was revealed that AtGAT1 mediates H -dependent, high affinity expressed in this host [74]. A recombinant potato apyrase transport of high affinity γ-aminobutyric acid (GABA) was expressed and purified in the hyperglycosylated form at and GABA-related compounds. Properties of this protein 1 mg/L protein concentration [75]. The catalytically active couldbeexaminedinmoredetailin Xenopus oocytes [60]. barley oxalate oxidase, HvOXO was produced with a yield of Heterologous expression of AtTIP2;1 and AtTIP2;3 from 50 mg/L culture and biochemically characterized [76]. Arabidopsis in both ammonium uptake-defective yeast and High-level expression of wheat germin/oxalate oxidase oocytes indicated that these TIPs transport both ammonium was achieved in P. pastorisas an α-mating factor signal pep- and methyl-ammonium in addition to water and urea tide fusion to increase secretion of the protein of interest into [61]. The kinetic characteristics of the sorbitol transporters, the culture medium. Approximately 1 g (4×10 U) of TaOXO PmPLT1, and PmPLT2 from common plantain (Plantago was produced in 5 L fermentation cultures following 8 days of major) were investigated by functional expression in S. methanol induction, demonstrating the possibility of large- cerevisiae and in Xenopus oocytess. In the yeast system, scale production of oxalate oxidase for biotechnological both proteins were characterized as low-affinity and low- applications. Glycosylation of the recombinant protein was specificity polyol symporters. These data were confirmed in evidencedbymassspectrometry[77]. Another application the Xenopus system, where PmPLT1 was analyzed in detail using P. pastoris is the expression of the α-subunit of and characterized as an H symporter [62]. heterotrimeric G-proteins, GPA1, from Arabidopsis. Several The major disadvantages of using S. cerevisiae mutants attempts had previously failed to produce this protein in in transporter studies are the hyperpolarization of the mem- E. coli, whereas in the yeast system the protein could be brane, mislocalization of membrane proteins and recruit- expressed with a his -tag and purified by affinity chromatog- + + ment of non-K -transporters into K -transporters [63]. raphy with a yield up to 20 mg from 700 mL culture [78]. Several allergens including, Cyn d 1 from Bermuda grass, Pichia Pastoris. P. pastoris, methylotrophic yeast, is consid- Bla g 4 from German cockroach,Amb a6from Ambrosia artemisiifolia, andOlee1from Olea europaea have also been ered a valuable tool for high yield heterologous expression of various proteins. The possibility of obtaining posttransla- produced in P. pastoris (see list in 64). tional modifications, high level expression of foreign proteins This system was also used for the expression of a number of plant lectins such as Canavalia brasiliensis lectin (ConBr) in either intracellular or extracellular forms, simplicity of genetic manipulations, and availability of various P. [2] and the Nicotiana tabacum lectin [79]. In a recent pastoris strains and vectors make this expression system study, the low-affinity cation transporter (LCT1) from wheat highly popular [64]. Molecular manipulations such as gene was also expressed and functionally characterized using P. targeting, high frequency DNA transformation, and cloning pastoris [80]. for functional complementation are similar to those in S. cerevisiae [64]. Tightly regulated promoters, easy integration 3.2.2. Insect Cells. Baculoviruses have been used for the of heterologous DNA into the host chromosome and the synthesis of a wide variety of eukaryotic recombinant capacity to generate more posttranslational modifications proteins in insect cells. In this expression system one of make P. pastoris the preferred system compared to S. the nonessential viral genes is replaced with the target cerevisiae. protein through homologous recombination. The resulting The wide use of P. pastoris expression system for reco- recombinant baculovirus is used to infect cultured insect mbinant plant proteins can be seen from recent reviews cells and the heterologous genes can be expressed under the [64, 65]. P. pastoris is particularly well suited for studying control of the extremely strong pPolh, polyhedron promoter plant enzymes since glycosylation of the foreign proteins in the late phase of infection. is expected to be closer to that in plants [66, 67]and The most common baculovirus used for expression glycosylated proteins have shorter glycosyl chains in P. studies is Autographa californica multiple capsid nucle- pastoris than in S. cerevisiae [68]. This expression system has opolyhedrovirus (AcMNPV) and the most frequently used the potential to produce high levels of recombinant proteins host insects are Spodoptera frugiperda and Trichoplusia ni. [67], up to 400 mg/L of culture [69]. Several plant enzymes This expression system produces high levels of recombinant have been produced in Pichia. Two examples are cytosolic proteins which are soluble, post-translationally modified, expression of nitrate reductase from spinach and corn at biologically active, and functional [81]. The virus is not high levels needed for detailed biochemical studies [69]and pathogenic to vertebrates or plants. The main drawback of expression of a sweet potato invertase in milligram quantities this system over the bacterial and yeast systems lies in the [70]. Enzymatic activity of the membrane-bound α1,6- noncontinuous expression of the heterologous gene; every galactosyltransferase was shown through overexpression in round of protein production needs reinfection [3]. P. pastoris [71]. The hypothesis that α-xylosyltransferase is This heterologous expression system is mainly used to involved in xyloglucan biosynthesis was tested by overex- investigate enzymatic mechanisms in plants. The most recent pressing the corresponding genes and identifying the gene examples include the Arabidopsis reductase isoforms, AR1 product that displayed activity [72]. P. pastoris has been used and AR2 [82], peroxisomal short-chain acyl-CoA oxidase A for production of a number of glycosyltransferases involved [83], cyclin-dependent kinase A [84], NADH-cytochrome in the biosynthesis of N-and O-linked oligosaccharides b5 reductase [85], geranylgeranyltransferase-I [86], acyl-CoA 10 International Journal of Plant Genomics synthetase [87], homogentisate phytyltransferase [88], (+)- transporters [110, 111], polyol transporters [112, 113], inos- abscisic acid 8’-hydroxylase [89], β1,2-xylosyltransferase itol transporters [114, 115], an amino acid transporter [116], [90], tobacco ethylene-inducing xylanase [91], and barley a cation–Cl-cotransporter [117], and an anion-selective ADP-glucose pyrophoshorylase [92]. The overall yield of transporter [118]in Xenopus oocytes were investigated. heterelogous proteins obtained with this system is usually Cases where channel proteins expressed in oocytes were not lower than with P. pastoris. functional have also been reported. These include the K Baculovirus-infected insect cells have been used as an channels AKT1 from Arabidopsis [93, 119, 120], TaAKT1 alternative system to Xenopus oocytes for expression and from wheat [121], DKT1 from carrot [122], and OsAKT1 characterization of plant channel proteins. Several channel from rice [123]. The causes for the lack of function of these proteins which were not functional in oocytes could be recombinant proteins are not clear. characterized in baculovirus-infected insect cells such as the Several studies have used expression of a wild type and K channel proteins AKT1 [93], KAT1 [94], KCO1 [95]from its mutant forms in Xenopus oocytes to confirm the in Arabidopsis, and KST1 [96]and SKT1 [97]frompotato. vivo functions of plant proteins, especially transporters and To investigate the interaction between AUX1 and its plasma membrane intrinsic proteins (PIPs or aquaporins). transport substrate indole-3-acetic acid (IAA) from Ara- To demonstrate whether or not the plant K channels form bidopsis, an epitope-tagged version of AUX1 was expressed multimers, the wild type and a mutant were coexpressed in at high levels in a baculovirus expression system and suitable Xenopus oocytes [120]. Coexpression of tomato ammonium membrane fragments were prepared from baculovirus- transporter (LeAMT1;1) and its mutant in Xenopus oocytes infected insect cells for direct measurement of IAA binding inhibited ammonium transport, suggesting homooligomer- to AUX1. AUX1-IAA interactions were determined using a ization [105]. In another study, the role of phosphorylation radio-ligand binding assay to confirm that AUX1 was able to in the water channel activity of wild-type and mutant bind IAA with an affinity (Kd) of 2.6 mM, comparable with ZmPIP2;1 was studied in Xenopus oocytes [124]. estimates of the Km for IAA transport [98]. In recent studies, the Xenopus oocyte expression system The main disadvantages of using baculovirus-infected was used to investigate structure-function relationships. In insect cells are difficulties in constructing the expression one example, differences in the function of two cation vectors, requirements for more complex laboratory facilities transporters, wheat HKT1 and Arabidopsis AtHKT1, were and skills, and the short expression periods after infection. investigated using a series of AtHKT1/HKT1 chimeras with point mutations [103]. 3.2.3. Xenopus Laevis Oocytes. The oocytes of the South African clawed frog, Xenopus laevis, are also used for 4. Conclusions heterelogous expression of eukaryotic genes. The mRNA for the target protein, introduced by microinjection into the Heterologous expression of plant genes in other host cytoplasm, is translated and the protein is posttranslationally organisms has two main applications: (1) overexpression modified by the oocyte [99]. Direct injections of DNA into of the encoded protein, for biochemical and biophysical the nucleus are also possible, but the manipulations are characterization and (2) expression of foreign genes for difficult as the nucleus can easily be damaged in the process. determination of the function of the encoded protein Investigations on membrane transport proteins can be by complementing in a mutant host. Overexpression of readily performed on oocytes where techniques for electro- recombinant proteins is usually carried out with a cleavable physiological measurements are well established. Although, tag to simplify purification in large quantities. In contrast, a high proportion of cells express the foreign gene after complementation studies are carried out in null mutants to injection variations in the quality of oocytes and in the ability restore a missing activity in vivo. of individual cells to produce the heterelogous protein can Decisions on which expression vectors to use and the cause problems. Oocytes are not suitable for preparing large choice of the expression host depend on the particular application. In general E.coli is the first choice as host because quantities of proteins and the short expression period often leads to technical difficulties. The system can also not be of its simplicity, availability of expression vectors, cost effec- sustained over long periods of time and is not suitable for tiveness, and availability of extensive genetic information on stable expression [99, 100]. this host. Alternative expression systems are used only if Xenopus oocytes have,however,providedapowerful the recombinant protein is inactive due to lack of essential heterologous expression system for animal as well as plant posttranslational modifications and when detailed studies genes.The possibility of using Xenopus oocytes as heterol- on the recombinant protein function are planned. Yeast ogous expression systems for the identification of plant systems have the advantage of ease of manipulation and short transporters was first demonstrated by the expression of generation time. S. cerevisiae has been extensively used for the H /glucose transporter STP1 from Arabidopsis [101]. It functional complementation, biochemical, and electrophysi- has, since, been mainly used for production of transporters olagical characterization of plant membrane and transporter including potassium channels, H /hexose cotransporters, proteins. P. pastoris is the preferred host for overexpression aquaporins, and chloride channels [99]. In addition, func- of several plant enzymes. Baculovirus-mediated insect cell tional expression of a nitrate transporter [102],aK /Na expression offers the possibility for detailed investigations of transporter [39, 103, 104], ammonium transporters [105, plant enzymes and transporters. The oocyte from Xenopus 106], sucrose transporters [107–109], Al-activated malate laevis is often used for monitoring activity and biochemical International Journal of Plant Genomics 11 and electrophysiological characterization of plant plasma prospects,” Current Opinion in Biotechnology, vol. 16, no. 5, pp. 538–545, 2005. membrane transporter and pump proteins. [12] A. Shokri, A. M. Sanden, ´ and G. Larsson, “Cell and Heterologous expression is a powerful tool for functional process design for targeting of recombinant protein into the and biochemical analyses of genes and gene families isolated culture medium of Escherichia coli,” Applied Microbiology and from various organisms. It is particularly important for Biotechnology, vol. 60, no. 6, pp. 654–664, 2003. plants where the whole genome sequence is not available. [13] F. Baneyx and M. Mujacic, “Recombinant protein folding and This system will also provide denovo analysis. Its limita- misfolding in Escherichia coli,” Nature Biotechnology, vol. 22, tions, however, should be kept in mind, especially when no. 11, pp. 1399–1408, 2004. interpreting the results in terms of the native structure and [14] J. H. Choi and S. Y. Lee, “Secretory and extracellular function of proteins. Major problems arise from misfolding production of recombinant proteins using Escherichia coli,” and mislocalization of recombinant proteins in foreign hosts. Applied Microbiology and Biotechnology,vol. 64, no.5,pp. Strategies developed to avoid misfolding of recombinant 625–635, 2004. [15] Y. Kurokawa, H. Yanagi, and T. Yura, “Overproduction of proteins include expression in periplasmic space, expression bacterial protein disulfide isomerase (DsbC) and its modu- with a tag, and utilization of different hosts. Mislocalization, lator (DsbD) markedly enhances periplasmic production of on the other hand, may occur because the recombinant human nerve growth factor in Escherichia coli,” The Journal of protein may take over the function of the missing host Biological Chemistry, vol. 276, no. 17, pp. 14393–14399, 2001. protein [125]. Conclusions on function need to be tested in [16] S. Sahdev, S. K. Khattar, and K. S. Saini, “Production of active alternative hosts and eventually in the plant itself. eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies,” Molecular References and Cellular Biochemistry, vol. 307, no. 1-2, pp. 249–264, [1] Y. Yan, J. Chen, and J. Li, “Overexpression of a small 2008. [17] F. J. M. Mergulha ˜o,D.K.Summers,and G. A. Monteiro, medicinal peptide from ginseng in the yeast Pichia pastoris,” Protein Expression and Purification, vol. 29, no. 2, pp. 161– “Recombinant protein secretion in Escherichia coli,” Biotech- 166, 2003. nology Advances, vol. 23, no. 3, pp. 177–202, 2005. [18] Y. Xu, D. Lewis, and C. P. Chou, “Effect of folding factors [2] W. M. Bezerra, C. P. Carvalho, R. A. Moreira, and T. B. Grangeiro, “Establishment of a heterologous system for the in rescuing unstable heterologous lipase B to enhance its overexpression in the periplasm of Escherichia coli,” Applied expression of Canavalia brasiliensis lectin: a model for the study of protein splicing,” Genetics and Molecular Research, Microbiology and Biotechnology, vol. 79, no. 6, pp. 1035–1044, vol. 5, no. 1, pp. 216–223, 2006. [3] J. Yin, G. Li, X. Ren, and G. Herrler, “Select what you need: a [19] P. H. Bessette, F. Aslund, J. Beckwith, and G. Georgiou, “Efficient folding of proteins with multiple disulfide bonds comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes,” Journal in the Escherichia coli cytoplasm,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, of Biotechnology, vol. 127, no. 3, pp. 335–347, 2007. [4] K. Terpe, “Overview of tag protein fusions: from molecular no. 24, pp. 13703–13708, 1999. [20] W. A. Prinz, F. Aslund, A. Holmgren, and J. Beckwith, and biochemical fundamentals to commercial systems,” Applied Microbiology and Biotechnology,vol. 60, no.5,pp. “The role of the thioredoxin and glutaredoxin pathways 523–533, 2003. in reducing protein disulfide bonds in the Escherichia coli cytoplasm,” The Journal of Biological Chemistry, vol. 272, no. [5] R. B. Kapust and D. S. Waugh, “Escherichia coli maltose- binding protein is uncommonly effective at promoting the 25, pp. 15661–15667, 1997. [21] P. Cassland, S. Larsson, N.-O. Nilvebrant, and L. J. Jonsson, ¨ solubility of polypeptides to which it is fused,” Protein Science, vol. 8, no. 8, pp. 1668–1674, 1999. “Heterologous expression of barley and wheat oxalate oxi- dase in an E. coli trxB gor double mutant,” Journal of [6] F. Baneyx, “Recombinant protein expression in Escherichia coli,” Current Opinion in Biotechnology,vol. 10, no.5,pp. Biotechnology, vol. 109, no. 1-2, pp. 53–62, 2004. [22] S. S. Newton andJ.G.Duman,“An osmotin-like cryopro- 411–421, 1999. [7] W. B. Frommer and O. Ninnemann, “Heterologous expres- tective protein from the bittersweet nightshade Solanum dulcamara,” Plant Molecular Biology, vol. 44, no. 5, pp. 581– sion of genes in bacterial, fungal, animal, and plant cells,” Annual Review of Plant Physiology and Plant Molecular 589, 2000. [23] X. Hu and A. S. N. Reddy, “Cloning and expression of a PR5- Biology, vol. 46, pp. 419–444, 1995. [8] A.Lueking,C.Holz,C.Gotthold,H.Lehrach,and D. Cahill, like protein from Arabidopsis: inhibition of fungal growth by “A system for dual protein expression in Pichia pastoris and bacterially expressed protein,” Plant Molecular Biology, vol. 34, no. 6, pp. 949–959, 1997. Escherichia coli,” Protein Expression and Purification, vol. 20, no. 3, pp. 372–378, 2000. [24] M. de A Campos, M. S. Silva, C. P. Magalhaes, ˜ et al., “Expression in Escherichia coli, purification, refolding and [9] G. Fuh, M. G. Mulkerrin, S. Bass, et al., “The human growth hormone receptor. Secretion from Escherichia coli antifungal activity of an osmotin from Solanum nigrum,” Microbial Cell Factories, vol. 7, pp. 7–17, 2008. and disulfide bonding pattern of the extracellular binding domain,” The Journal of Biological Chemistry, vol. 265, no. 6, [25] A. J. Afzal and D. A. Lightfoot, “Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded pp. 3111–3115, 1990. [10] C. Wulfing ¨ and A. Pluc ¨ kthun, “Protein folding in the from Escherichia coli inclusion bodies: preparation for a functional analysis,” Protein Expression and Purification, vol. periplasm of Escherichia coli,” Molecular Microbiology, vol. 12, no. 5, pp. 685–692, 1994. 53, no. 2, pp. 346–355, 2007. [26] P. K. Carvajal-Vallejos, A. Campos, P. Fuentes-Prior, et al., [11] G. Georgiou and L. Segatori, “Preparative expression of secreted proteins in bacteria: status report and future “Purification and in vitro refolding of maize chloroplast 12 International Journal of Plant Genomics transglutaminase over-expressed in Escherichia coli,” Biotech- [41] G. Mir, J. Domenec ` h, G. Huguet, et al., “A plant type 2 nology Letters, vol. 29, no. 8, pp. 1255–1262, 2007. metallothionein (MT) from cork tissue responds to oxidative [27] J. K. Jha, M. K. Maiti, A. Bhattacharjee, A. Basu, P. C. Sen, stress,” Journal of Experimental Botany, vol. 55, no. 408, pp. and S. K. Sen, “Cloning and functional expression of an acyl- 2483–2493, 2004. ACP thioesterase FatB type from Diploknema (Madhuca) [42] F. Dede,G.Dinler,and Z. Sayers,“3D Macromolecular butyracea seeds in Escherichia coli,” Plant Physiology and structure analyses: applications in plant proteins,” in Proceed- Biochemistry, vol. 44, no. 11-12, pp. 645–655, 2006. ings of the NATO Advanced Research Workshop, pp. 135–146, [28] J. M. Jez, R. E. Cahoon, and S. Chen, “Arabidopsis Springer, 2006. thaliana glutamate-cysteine ligase. Functional properties, [43] F. Yesilirmak, Biophysical and functional characterization of kinetic mechanism, and regulation of activity,” The Journal of wheat metallothionein at molecular level, Ph.D. thesis, Sabanci Biological Chemistry, vol. 279, no. 32, pp. 33463–33470, 2004. University, Istanbul, Turkey, 2008. [29] X. Sun, N. Frearson, C. Kirk, et al., “An E. coli expression [44] Y. Zhan, X. Song, and G. W. Zhou, “Structural analysis system optimized for DELLA proteins,” Protein Expression of regulatory protein domains using GST-fusion proteins,” and Purification, vol. 58, no. 1, pp. 168–174, 2008. Gene, vol. 281, no. 1-2, pp. 1–9, 2001. [30] N. Uozumi, T. Nakamura, J. I. Schroeder, and S. Muto, [45] M. Schmidt and D. R. Hoffman, “Expression systems for “Determination of transmembrane topology of an inward- production of recombinant allergens,” International Archives rectifying potassium channel from Arabidopsis thaliana based of Allergy and Immunology, vol. 128, no. 4, pp. 264–270, 2002. on functional expression in Escherichia coli,” Proceedings of [46] I. Dreyer, C. Horeau, G. Lemaillet, et al., “Identification the National Academy of Sciences of the United States of and characterization of plant transporters using heterologous America, vol. 95, no. 17, pp. 9773–9778, 1998. expression systems,” Journal of Experimental Botany, vol. 50, [31] E. J. Kim, J. M. Kwak,N.Uozumi, andJ.I.Schroeder, pp. 1073–1087, 1999. “AtKUP1:an Arabidopsis gene encoding high-affinity potas- [47] A. Bertl, J. A. Anderson, C. L. Slayman, and R. F. Gaber, sium transport activity,” The Plant Cell, vol. 10, no. 1, pp. 51– “Use of Saccharomyces cerevisiae for patch-clamp analysis 62, 1998. of heterologous membrane proteins: characterization of [32] N. Uozumi, E. J. Kim, F. Rubio, et al., “The Arabidopsis HKT1 Kat1, an inward-rectifying K channel from Arabidopsis gene homolog mediates inward Na currents in Xenopus thaliana, and comparison with endogeneous yeast channels laevis oocytes and Na uptake in Saccharomyces cerevisiae,” and carriers,” Proceedings of the National Academy of Sciences Plant Physiology, vol. 122, no. 4, pp. 1249–1259, 2000. of the United States of America, vol. 92, no. 7, pp. 2701–2705, [33] D. J. Fairbairn, W. Liu, D. P. Schachtman, S. Gomez-Gallego, 1995. S. R. Day, and R. D. Teasdale, “Characterisation of two [48] U. S. Muchhal, J. M. Pardo, and K. G. Raghothama, distinct HKT1-like potassium transporters from Eucalyptus “Phosphate transporters from the higher plant Arabidopsis camaldulensis,” Plant Molecular Biology, vol. 43, no. 4, pp. thaliana,” Proceedings of the National Academy of Sciences of 515–525, 2000. the United States of America, vol. 93, no. 19, pp. 10519–10523, [34] J. Tjaden, C. Schwopp ¨ e, T. Mohlmann, ¨ P. W. Quick, and 1996. H. E. Neuhaus, “Expression of a plastidic ATP/ADP trans- [49] G. E. Santa-Mar´ ıa, F. Rubio, J. Dubcovsky, and A. Rodr´ ıguez- porter gene in Escherichia coli leads to a functional adenine Navarro, “The HAK1 gene of barley is a member of a nucleotide transport system in the bacterial cytoplasmic large gene family and encodes a high-affinity potassium membrane,” The Journal of Biological Chemistry, vol. 273, no. transporter,” The Plant Cell, vol. 9, no. 12, pp. 2281–2289, 16, pp. 9630–9636, 1998. 1997. [35] B. Garciadeblas, B. Benito, and A. Rodr´ ıguez-Navarro, [50] F. J. Quintero and M. R. Blatt, “A new family of K “Molecular cloning and functional expression in bacteria of transporters from Arabidopsis that are conserved across the potassium transporters CnHAK1 and CnHAK2 of the phyla,” FEBS Letters, vol. 415, no. 2, pp. 206–211, 1997. seagrass Cymodocea nodosa,” Plant Molecular Biology, vol. 50, [51] H.-H. Fu and S. Luan, “AtKUP1: a dual-affinity K trans- no. 4-5, pp. 623–633, 2002. porter from arabidopsis,” The Plant Cell,vol. 10, no.1,pp. [36] J. Jeong, S. Suh, C. Guan, et al., “A nodule-specific dicar- 63–73, 1998. boxylate transporter from alder is a member of the peptide [52] D. P. Schachtman and J. I. Schroeder, “Structure and transporter family,” Plant Physiology, vol. 134, no. 3, pp. 969– transport mechanism of a high-affinity potassium uptake 978, 2004. transporter from higher plants,” Nature, vol. 370, no. 6491, [37] K. Bilecen, U. H. Ozturk, A. D. Duru, et al., “Triticum pp. 655–658, 1994. durum metallothionein: isolation of the gene and structural [53] F. Rubio, W. Gassmann, and J. I. Schroeder, “Sodium-driven characterization of the protein using solution scattering and potassium uptake by the plant potassium transporter HKT1 molecular modeling,” The Journal of Biological Chemistry, and mutations conferring salt tolerance,” Science, vol. 270, vol. 280, no. 14, pp. 13701–13711, 2005. no. 5242, pp. 1660–1663, 1995. [38] R. C. Foley, Z. M. Liang, and K. B. Singh, “Analysis of type [54] J. R. Howarth, P. Fourcroy, J.-C. Davidian, F. W. Smith, and 1 metallothionein cDNAs in Vicia faba,” Plant Molecular M. J. Hawkesford, “Cloning of two contrasting high-affinity Biology, vol. 33, no. 4, pp. 583–591, 1997. sulfate transporters from tomato induced by low sulfate [39] A. Murphy, J. Zhou, P. B. Goldsbrough, and L. Taiz, “Purifi- and infection by the vascular pathogen Verticillium dahliae,” cation and immunological identification of metallothioneins Planta, vol. 218, no. 1, pp. 58–64, 2003. 1and 2from Arabidopsis thaliana,” Plant Physiology, vol. 113, [55] V. Sancenon, ´ S. Puig, H. Mira, D. J. Thiele, and L. Penar ˜ rubia, no. 4, pp. 1293–1301, 1997. “Identification of a copper transporter family in Arabidopsis [40] S. N. A. Abdullah,S.C.Cheah,and D. J. Murphy,“Isolation thaliana,” Plant Molecular Biology, vol. 51, no. 4, pp. 577–587, and characterisation of two divergent type 3 metalloth- 2003. ioneins from oil palm, Elaeis guineensis,” Plant Physiology and [56] D. Dietrich, U. Hammes, K. Thor, et al., “AtPTR1, a Biochemistry, vol. 40, no. 3, pp. 255–263, 2002. plasma membrane peptide transporter expressed during seed International Journal of Plant Genomics 13 germination and in vascular tissue of Arabidopsis,” The Plant matrix polysaccharide biosynthesis,” The Plant Journal, vol. Journal, vol. 40, no. 4, pp. 488–499, 2004. 19, no. 6, pp. 691–697, 1999. [72] A. Faik,N.J.Price,N.V.Raikhel,and K. Keegstra,“An [57] L. Maresova and H. Sychrova, “Arabidopsis thaliana CHX17 Arabidopsis gene encoding an α-xylosyltransferase involved gene complements the kha1 deletion phenotypes in Saccha- in xyloglucan biosynthesis,” Proceedings of the National romyces cerevisiae,” Yeast, vol. 23, no. 16, pp. 1167–1171, Academy of Sciences of the United States of America, vol. 99, no. 11, pp. 7797–7802, 2002. [58] M. A. Hayes, C. Davies, and I. B. Dry, “Isolation, functional [73] M. Bencur ´ ova, ´ D. Rendic, ´ G. Fabini, E.-M. Kopecky, F. characterization, and expression analysis of grapevine (Vitis Altmann, and I. B. H. Wilson, “Expression of eukaryotic vinifera L.) hexose transporters: differential roles in sink and glycosyltransferases in the yeast Pichia pastoris,” Biochimie, source tissues,” Journal of Experimental Botany, vol. 58, no. 8, vol. 85, no. 3-4, pp. 413–422, 2003. pp. 1985–1997, 2007. [74] R.-H. Fu, A.-Y. Wang, Y.-C. Wang, and H.-Y. Sung, “A cDNA [59] S. Schneider, A. Schneidereit, P. Udvardi, et al., “Arabidopsis encoding vacuolar type β-D-fructofuranosidase (Osβfruct3) Inositol Transporter2 mediates H symport of different inos- of rice and its expression in Pichia pastoris,” Biotechnology itol epimers and derivatives across the plasma membrane,” Letters, vol. 25, no. 18, pp. 1525–1530, 2003. Plant Physiology, vol. 145, no. 4, pp. 1395–1407, 2007. [75] N. Nourizad, M. Ehn, B. Gharizadeh, S. Hober, and P. Nyren, ´ [60] A. Meyer, S. Eskandari, S. Grallath, and D. Rentsch, “AtGAT1, “Methylotrophic yeast Pichia pastoris as a host for production a high affinity transporter for γ-aminobutyric acid in of ATP-diphosphohydrolase (apyrase) from potato tubers Arabidopsis thaliana,” The Journal of Biological Chemistry, vol. (Solanum tuberosum),” Protein Expression and Purification, 281, no. 11, pp. 7197–7204, 2006. vol. 27, no. 2, pp. 229–237, 2003. [61] D. Loque, ´ U. Ludewig, L. Yuan, and N. von Wiren, ´ “Tono- [76] M. M. Whittaker and J. W. Whittaker, “Characterization plast intrinsic proteins AtTIP2;1 and AtTIP2;3 facilitate NH of recombinant barley oxalate oxidase expressed by Pichia transport into the vacuole,” Plant Physiology, vol. 137, no. 2, pastoris,” Journal of Biological Inorganic Chemistry, vol. 7, no. pp. 671–680, 2005. 1-2, pp. 136–145, 2002. [62] M. Ramsperger-Gleixner, D. Geiger, R. Hedrich, and N. [77] H.-Y. Pan, M. M. Whittaker, R. Bouveret, A. Berna, F. Bernier, Sauer, “Differential expression of sucrose transporter and and J. W. Whittaker, “Characterization of wheat germin polyol transporter genes during maturation of common (oxalate oxidase) expressed by Pichia pastoris,” Biochemical plantain companion cells,” Plant Physiology, vol. 134, no. 1, and Biophysical Research Communications, vol. 356, no. 4, pp. pp. 147–160, 2004. 925–929, 2007. [63] R. Madrid, M. J. Gomez, ´ J. Ramos, and A. Rodr´ ıguez- [78] B. Kaplan, S. Tunca, and Z. Sayers, “Expression of A. thaliana Navarro, “Ectopic potassium uptake in trk1 trk2 mutants G protein alpha subunit in P. pastoris,” FEBS Journal, vol. 272, of Saccharomyces cerevisiae correlates with a highly hyper- supplement 1, pp. 1–11, 2005. polarized membrane potential,” The Journal of Biological [79] N. Lannoo, W. Vervecken, P. Proost, P. Rouge, ´ and E. J. M. Chemistry, vol. 273, no. 24, pp. 14838–14844, 1998. Van Damme, “Expression of the nucleocytoplasmic tobacco [64] J. L. Cereghino and J. M. Cregg, “Heterologous protein lectin in the yeast Pichia pastoris,” Protein Expression and expression in the methylotrophic yeast Pichia pastoris,” FEMS Purification, vol. 53, no. 2, pp. 275–282, 2007. Microbiology Reviews, vol. 24, no. 1, pp. 45–66, 2000. [80] E. Diatloff,B.G.Forde,and S. K. Roberts, “Expression and transport characterisation of the wheat low-affinity cation [65] S. Macauley-Patrick,M.L.Fazenda,B.McNeil, andL.M. transporter (LCT1) in the methylotrophic yeast Pichia pas- Harvey, “Heterologous protein production using the Pichia toris,” Biochemical and Biophysical Research Communications, pastoris expression system,” Yeast, vol. 22, no. 4, pp. 249–270, vol. 344, no. 3, pp. 807–813, 2006. [81] D. L. Andrews, B. Beames, M. D. Summers, and W. D. Park, [66] L. S. Grinna and J. F. Tschopp, “Size distribution and general “Characterization of the lipid acyl hydrolase activity of the structural features of N-linked oligosaccharides from the major potato (Solanum tuberosum) tuber protein, patatin, by methylotrophic yeast, Pichia pastoris,” Yeast,vol. 5, no.2,pp. cloning and abundant expression in a baculovirus vector,” 107–115, 1989. Biochemical Journal, vol. 252, no. 1, pp. 199–206, 1988. [67] J. M. Cregg, T. S. Vedvick, and W. C. Raschke, “Recent [82] M. Mizutani and D. Ohta, “Two isoforms of NAPDH: advances in the expression of foreign genes in Pichia pastoris,” cytochrome p450 reductase in Arabidopsis thaliana gene Nature Biotechnology, vol. 11, no. 8, pp. 905–910, 1993. structure, heterologous expression in insect cells, and differ- [68] R. K. Bretthauer and F. J. Castellino, “Glycosylation of ential regulation,” Plant Physiology, vol. 116, no. 1, pp. 357– Pichia pastoris-derived proteins,” Biotechnology and Applied 367, 1998. Biochemistry, vol. 30, no. 3, pp. 193–200, 1999. [83] H. Hayashi, L. De Bellis, A. Ciurli, M. Kondo, M. Hayashi, [69] J. A. Mertens, N. Shiraishi, and W. H. Campbell, “Recom- and M. Nishimura, “A novel Acyl-CoA oxidase that can binant expression of molybdenum reductase fragments of oxidize short-chain Acyl-CoA in plant peroxisomes,” The plant nitrate reductase at high levels in Pichia pastoris,” Plant Journal of Biological Chemistry, vol. 274, no. 18, pp. 12715– Physiology, vol. 123, no. 2, pp. 743–756, 2000. 12721, 1999. [70] W.-C. Huang, A.-Y. Wang, L.-T. Wang, and H.-Y. Sung, [84] H. Harashima, A. Shinmyo, and M. Sekine, “Phosphoryla- “Expression and characterization of sweet potato invertase in tion of threonine 161 in plant cyclin-dependent kinase A Pichia pastoris,” Journal of Agricultural and Food Chemistry, is required for cell division by activation of its associated vol. 51, no. 5, pp. 1494–1499, 2003. kinase,” The Plant Journal, vol. 52, no. 3, pp. 435–448, 2007. [71] M. E. Edwards, C. A. Dickson, S. Chengappa, C. Sidebottom, [85] M. Fukuchi-Mizutani, M. Mizutani, Y. Tanaka, T. Kusumi, M. J. Gidley, and J. S. G. Reid, “Molecular characterisation and D. Ohta, “Microsomal electron transfer in higher plants: of a membrane-bound galactosyltransferase of plant cell wall cloning and heterologous expression of NADH-cytochrome 14 International Journal of Plant Genomics b reductase from Arabidopsis,” Plant Physiology, vol. 119, no. Nutrition and Food Research, vol. 49, no. 3, pp. 228–234, 1, pp. 353–361, 1999. 2005. [86] D. Caldelari, H. Sternberg, M. Rodr´ ıguez-Concepcion, ´ W. [101] K. J. Boorer, B. G. Forde, R. A. Leigh, and A. J. Miller, “Func- Gruissem, and S. Yalovsky, “Efficient prenylation by a plant tional expression of a plant plasma membrane transporter in geranylgeranyltransferase-I requires a functional Caal box Xenopus oocytes,” FEBS Letters, vol. 302, no. 2, pp. 166–168, motif and a proximal polybasic domain,” Plant Physiology, 1992. vol. 126, no. 4, pp. 1416–1429, 2001. [102] M. Orsel, F. Chopin, O. Leleu, et al., “Characterization [87] H. Hayashi, L. De Bellis, Y. Hayashi, et al., “Molecular char- of a two-component high-affinity nitrate uptake system acterization of an Arabidopsis acyl-coenzyme a synthetase in Arabidopsis. Physiology and protein-protein interaction,” localized on glyoxysomal membranes,” Plant Physiology, vol. Plant Physiology, vol. 142, no. 3, pp. 1304–1317, 2006. 130, no. 4, pp. 2019–2026, 2002. [103] W. Liu, D. J. Fairbairn, R. J. Reid, and D. P. Schachtman, [88] B. Savidge, J. D. Weiss, Y.-H. H. Wong, et al., “Isolation and “Characterization of two HKT1 homologues from Eucalyptus characterization of homogentisate phytyltransferase genes camaldulensis that display intrinsic osmosensing capability,” from Synechocystis sp. PCC 6803 and Arabidopsis,” Plant Plant Physiology, vol. 127, no. 1, pp. 283–294, 2001. Physiology, vol. 129, no. 1, pp. 321–332, 2002. [104] P. Maser ¨ , Y. Hosoo, S. Goshima, et al., “Glycine residues [89] S. Saito, N. Hirai, C. Matsumoto, et al., “Arabidopsis in potassium channel-like selectivity filters determine potas- CYP707As encode (+)-abscisic acid 8 -hydroxylase, a key sium selectivity in four-loop-per-subunit HKT transporters enzyme in the oxidative catabolism of abscisic acid,” Plant from plants,” Proceedings of the National Academy of Sciences Physiology, vol. 134, no. 4, pp. 1439–1449, 2004. of the United States of America, vol. 99, no. 9, pp. 6428–6433, [90] S. Pagny, F. Bouissonnie, M. Sarkar, et al., “Structural 2002. requirements for Arabidopsis β1, 2-xylosyltransferase activ- [105] U. Ludewig, S. Wilken, B. Wu, et al., “Homo- and het- ity and targeting to the Golgi,” The Plant Journal, vol. 33, no. erooligomerization of ammonium transporter-1 NH uni- 1, pp. 189–203, 2003. porters,” The Journal of Biological Chemistry, vol. 278, no. 46, pp. 45603–45610, 2003. [91] N. Furman-Matarasso, E. Cohen, Q. Du, N. Chejanovsky, U. Hanania, and A. Avni, “A point mutation in the ethylene- [106] B. Neuhauser ¨ , M. Dynowski, M. Mayer, and U. Ludewig, inducing xylanase elicitor inhibits the β-1-4-endoxylanase “Regulation of NH transport by essential cross talk between activity but not the elicitation activity,” Plant Physiology, vol. AMT monomers through the carboxyl tails,” Plant Physiol- 121, no. 2, pp. 345–351, 1999. ogy, vol. 143, no. 4, pp. 1651–1659, 2007. [92] D. N. P. Doan, H. Rudi, and O.-A. Olsen, “The allosterically [107] D. Chandran, A. Reinders, and J. M. Ward, “Substrate unregulated isoform of ADP-glucose pyrophosphorylase specificity of the Arabidopsis thaliana sucrose transporter from barley endosperm is the most likely source of ADP- AtSUC2,” The Journal of Biological Chemistry, vol. 278, no. glucose incorporated into endosperm starch,” Plant Physiol- 45, pp. 44320–44325, 2003. ogy, vol. 121, no. 3, pp. 965–975, 1999. [108] A. B. Sivitz, A. Reinders, M. E. Johnson, et al., “Arabidopsis [93] F. Gaymard, M. Cerutti, C. Horeau, et al., “The bac- sucrose transporter AtSUC9. High-affinity transport activity, ulovirus/insect cell system as an alternative to Xenopus intragenic control of expression, and early flowering mutant oocytes. First characterization of the AKT1 K channel from phenotype,” Plant Physiology, vol. 143, no. 1, pp. 188–198, Arabidopsis thaliana,” The Journal of Biological Chemistry, vol. 2007. 271, no. 37, pp. 22863–22870, 1996. [109] A. Reinders, A. B. Sivitz, C. G. Starker, J. S. Gantt, and J. [94] I. Marten,F.Gaymard,G.Lemaillet,J.-B. Thibaud, H. M. Ward, “Functional analysis of LjSUT4, a vacuolar sucrose Sentenac, and R. Hedrich, “Functional expression of the transporter from Lotus japonicus,” Plant Molecular Biology, plant K channel KAT1 in insect cells,” FEBS Letters, vol. 380, vol. 68, no. 3, pp. 289–299, 2008. no. 3, pp. 229–232, 1996. [110] T. Sasaki, Y. Yamamoto, B. Ezaki, et al., “A wheat gene [95] K. Czempinski, S. Zimmermann, T. Ehrhardt, and B. Muller ¨ - encoding an aluminum-activated malate transporter,” The Rober ¨ , “New structure and function in plant K channels: Plant Journal, vol. 37, no. 5, pp. 645–653, 2004. 2+ KCO1, an outward rectifier with a steep Ca dependency,” [111] A. Ligaba, M. Katsuhara, P. R. Ryan, M. Shibasaka, and The EMBO Journal, vol. 16, no. 10, pp. 2565–2575, 1997. H. Matsumoto, “The BnALMT1 and BnALMT2 genes from [96] T. Ehrhardt, S. Zimmermann, and B. Muller ¨ -Rober ¨ , “Associ- rape encode aluminum-activated malate transporters that ation of plant K (in) channels is mediated by conserved and enhance the aluminum resistance of plant cells,” Plant does not affect subunit assembly,” FEBS Letters, vol. 409, no. Physiology, vol. 142, no. 3, pp. 1294–1303, 2006. 2, pp. 166–170, 1997. [112] Y.-S. Klepek, D. Geiger, R. Stadler, et al., “Arabidopsis [97] S. Zimmermann, I. Talke, T. Ehrhardt, G. Nast, and B. POLYOL TRANSPORTERS, a new member of the monosac- Muller ¨ -Rob ¨ er, “Characterization of SKT1, an inwardly rec- charide transporter-like superfamily, mediates H -symport tifying potassium channel from potato, by heterologous in of numerous substrates, including myo-inositol, glycerol, and insect cells,” Plant Physiology, vol. 116, no. 3, pp. 879–890, ribose,” The Plant Cell, vol. 17, no. 1, pp. 204–218, 2005. [113] M. Ramsperger-Gleixner, D. Geiger, R. Hedrich, and N. [98] D. J. Carrier, N. T. A. Bakar, R. Swarup, et al., “The binding Sauer, “Differential expression of sucrose transporter and of auxin to the Arabidopsis auxin influx transporter AUX1,” polyol transporter genes during maturation of common Plant Physiology, vol. 148, no. 1, pp. 529–535, 2008. plantain companion cells,” Plant Physiology, vol. 134, no. 1, pp. 147–160, 2004. [99] A. J. Miller and J. J. Zhou, “Xenopus oocytes as an expression system for plant transporters,” Biochimica et Biophysica Acta, [114] S. Schneider, A. Schneidereit, K. R. Konrad, et al., “Arabidop- vol. 1465, no. 1-2, pp. 343–358, 2000. sis INOSITOL TRANSPORTER4 mediates high-affinity H [100] E. Sigel, “The Xenopus oocyte: system for the study of func- symport of myoinositol across the plasma membrane,” Plant tional expression and modulation of proteins,” Molecular Physiology, vol. 141, no. 2, pp. 567–577, 2006. International Journal of Plant Genomics 15 [115] S. Schneider, A. Schneidereit, P. Udvardi, et al., “Arabidop- for N-glycosylation of the pro-region,” The Journal of Biolog- sis INOSITOL TRANSPORTER2 mediates H symport of ical Chemistry, vol. 265, no. 27, pp. 16661–16666, 1990. different inositol epimers and derivatives across the plasma [131] K. L. Korth and C. S. Levings III, “Baculovirus expression of membrane,” Plant Physiology, vol. 145, no. 4, pp. 1395–1407, the maize mitochondrial protein URF13 confers insecticidal 2007. activity in cell cultures and larvae,” Proceedings of the [116] U. Z. Hammes, E. Nielsen, L. A. Honaas, C. G. Taylor, and D. National Academy of Sciences of the United States of America, vol. 90, no. 8, pp. 3388–3392, 1993. P. Schachtman, “AtCAT6, a sink-tissue-localized transporter for essential amino acids in Arabidopsis,” The Plant Journal, [132] J. Muschietti, L. Dircks, G. Vancanneyt, and S. McCormick, “LAT52 protein is essential for tomato pollen development: vol. 48, no. 3, pp. 414–426, 2006. pollen expressing antisense LAT52 RNA hydrates and germi- [117] J. M. Colmenero-Flores, G. Mart´ ınez, G. Gamba, et al., nates abnormally and cannot achieve fertilization,” The Plant “Identification and functional characterization of cation- Journal, vol. 6, no. 3, pp. 321–328, 1994. chloride cotransporters in plants,” The Plant Journal, vol. 50, [133] H. MacDonald, J. Henderson, R. M. Napier, M. A. Venis, no. 2, pp. 278–292, 2007. C. Hawes, and C. M. Lazarus, “Authentic processing and [118] M. A. Piner ˇ os, G. M. A. Canc¸ado,L.G.Maron,S.M.Lyi, targeting of active maize auxin-binding protein in the M. Menossi, and L. V. Kochian, “Not all ALMT1-type trans- baculovirus expression system,” Plant Physiology, vol. 105, porters mediate aluminum-activated organic acid responses: no. 4, pp. 1049–1057, 1994. the case of ZmALMT1—an anion-selective transporter,” The [134] J. M. Bauly, I. M. Sealy, H. Macdonald, et al., “Overexpression Plant Journal, vol. 53, no. 2, pp. 352–367, 2008. of auxin-binding protein enhances the sensitivity of guard [119] H. Sentenac, N. Bonneaud, M. Minet, et al., “Cloning and cells to auxin,” Plant Physiology, vol. 124, no. 3, pp. 1229– expression in yeast of a plant potassium ion transport 1238, 2000. system,” Science, vol. 256, no. 5057, pp. 663–665, 1992. + [135] H. Y. Meller Harel, V. Fontaine, H. Chen, I. M. Jones, and P. [120] I. Dreyer, S. Antunes, T. Hoshi, et al., “Plant K channel α- A. Millner, “Display of a maize cDNA library on baculovirus subunits assemble indiscriminately,” Biophysical Journal, vol. infected insect cells,” BMC Biotechnology, vol. 8, pp. 64–69, 72, no. 5, pp. 2143–2150, 1997. [121] P. H. Buschmann, R. Vaidyanathan, W. Gassmann, and J. + [136] M. Mizutani, D. Ohta, and R. Sato, “Isolation of a cDNA I. Schroeder, “Enhancement of Na uptake currents, time- and a genomic clone encoding cinnamate 4-hydroxylase + + dependent inward-rectifying K channel currents, and K + from Arabidopsis and its expression manner in planta,” Plant channel transcripts by K starvation in wheat root cells,” Physiology, vol. 113, no. 3, pp. 755–763, 1997. Plant Physiology, vol. 122, no. 4, pp. 1387–1397, 2000. [137] J.-P. Bouly, B. Giovani, A. Djamei, et al., “Novel ATP- [122] E. Formentin, S. Varotto, A. Costa, et al., “DKT1, a novel K binding and autophosphorylation activity associated with channel from carrot, forms functional heteromeric channels Arabidopsis and human cryptochrome-1,” European Journal with KDC1,” FEBS Letters, vol. 573, no. 1–3, pp. 61–67, 2004. of Biochemistry, vol. 270, no. 14, pp. 2921–2928, 2003. [123] I. Fuchs, S. Stolzle, ¨ N. Ivashikina, and R. Hedrich, “Rice K [138] H.-Y. Cho, T.-S. Tseng, E. Kaiserli, S. Sullivan, J. M. Christie, uptake channel OsAKT1 is sensitive to salt stress,” Planta, vol. and W. R. Briggs, “Physiological roles of the light, oxygen, 221, no. 2, pp. 212–221, 2005. or voltage domains of phototropin 1 and phototropin 2 in [124] V. Van Wilder, U. Miecielica, H. Degand, R. Derua, E. Arabidopsis,” Plant Physiology, vol. 143, no. 1, pp. 517–529, Waelkens, and F. Chaumont, “Maize plasma membrane aquaporins belonging to the PIP1 and PIP2 subgroups are in [139] A. Nagai, K. Suzuki, E. Ward, et al., “Overexpression of plant vivo phosphorylated,” Plant and Cell Physiology, vol. 49, no. histidinol dehydrogenase using a baculovirus expression 9, pp. 1364–1377, 2008. vector system,” Archives of Biochemistry and Biophysics, vol. [125] D. C. Bassham and N. V. Raikhel, “Plant cells are not just 295, no. 2, pp. 235–239, 1992. green yeast,” Plant Physiology, vol. 122, no. 4, pp. 999–1001, [140] A. Stapleton, J. K. Beetham, F. Pinot, et al., “Cloning and expression of soluble epoxide hydrolase from potato,” The [126] X. Dong, B. Tang, J. Li, Q. Xu, S. Fang, and Z. Hua, Plant Journal, vol. 6, no. 2, pp. 251–258, 1994. “Expression and purification of intact and functional soy- [141] S. Tada, M. Hatano, Y. Nakayama, et al., “Insect cell expres- bean (Glycine max) seed ferritin complex in Escherichia coli,” sion of recombinant imidazoleglycerolphosphate dehy- Journal of Microbiology and Biotechnology,vol. 18, no.2,pp. dratase of Arabidopsis and wheat and inhibition by triazole 299–307, 2008. herbicides,” Plant Physiology, vol. 109, no. 1, pp. 153–159, [127] J. Lin, R. Fido, P. Shewry, D. B. Archer, and M. J. C. Alcocer, “The expression and processing of two recombinant 2S [142] M. Schledz, S. Al-Babili, J. von Lintig, et al., “Phytoene albumins from soybean (Glycine max) in the yeast Pichia synthase from Narcissus pseudonarcissus: functional expres- pastoris,” Biochimica et Biophysica Acta, vol. 1698, no. 2, pp. sion, galactolipid requirement, topological distribution in 203–212, 2004. chromoplasts and induction during flowering,” The Plant [128] M. M. Bustos, V. A. Luckow, L. R. Griffing,M.D.Summers, Journal, vol. 10, no. 5, pp. 781–792, 1996. and T. C. Hall, “Expression, glycosylation and secretion of [143] S. Al-Babili, J. von Lintig, H. Haubruck, and P. Beyer, “A phaseolin in a baculovirus system,” Plant Molecular Biology, novel, soluble form of phytoene desaturase from Narcis- vol. 10, no. 6, pp. 475–488, 1988. sus pseudonarcissus chromoplasts is Hsp70-complexed and [129] R. Kunze and P. Starlinger, “The putative transposase of competent for flavinylation, membrane association and transposable element Ac from Zea mays L. interacts with enzymatic activation,” The Plant Journal,vol. 9, no.5,pp. subterminal sequences of Ac,” The EMBO Journal, vol. 8, no. 601–612, 1996. 11, pp. 3177–3185, 1989. [144] S. M. Allina, A. Pri-Hadash, D. A. Theilmann, B. E. Ellis, [130] T. Vernet, D. C. Tessier, C. Richardson, et al., “Secretion of and C. J. Douglas, “4-coumarate:coenzyme a ligase in hybrid functional papain precursor from insect cells. Requirement poplar. Properties of native enzymes, cDNA cloning, and 16 International Journal of Plant Genomics analysis of recombinant enzymes,” Plant Physiology, vol. 116, parasite Cuscuta reflexa,” Planta, vol. 213, no. 4, pp. 550–555, no. 2, pp. 743–754, 1998. 2001. [145] R. Pratelli, B. Lacombe, L. Torregrosa, et al., “A grapevine [159] S. Sakr, G. Alves, R. Morillon, et al., “Plasma membrane gene encoding a guard cell K channel displays developmen- aquaporins are involved in winter embolism recovery in tal regulation in the grapevine berry,” Plant Physiology, vol. walnut tree,” Plant Physiology, vol. 133, no. 2, pp. 630–641, 128, no. 2, pp. 564–577, 2002. 2003. [146] K. Philippar, K. Buc ¨ hsenschutz, ¨ M. Abshagen, et al., “The K [160] L.-H. Liu, U. Ludewig, B. Gassert, W. B. Frommer, and N. channel KZM1 mediates potassium uptake into the phloem von Wiren, ´ “Urea transport by nitrogen-regulated tonoplast and guard cells of the C grass Zea mays,” The Journal of intrinsic proteins in Arabidopsis,” Plant Physiology, vol. 133, Biological Chemistry, vol. 278, no. 19, pp. 16973–16981, 2003. no. 3, pp. 1220–1228, 2003. [147] K. Philippar, I. Fuchs, H. Luthen, ¨ et al., “Auxin-induced K [161] D. Loque, ´ U. Ludewig, L. Yuan, and N. von Wiren, ´ “Tono- channel expression represents an essential step in coleop- plast intrinsic proteins AtTIP2;1 and AtTIP2;3 facilitate NH tile growth and gravitropism,” Proceedings of the National transport into the vacuole,” Plant Physiology, vol. 137, no. 2, Academy of Sciences of the United States of America, vol. 96, pp. 671–680, 2005. no. 21, pp. 12186–12191, 1999. [162] R. Vera-Estrella, B. J. Barkla, H. J. Bohnert, and O. Pantoja, [148] S. Hartje, S. Zimmermann, D. Klonus, and B. Mueller- “Novel regulation of aquaporins during osmotic stress,” Plant Roeber, “Functional characterisation of LKT1, a K uptake Physiology, vol. 135, no. 4, pp. 2318–2329, 2004. channel from tomato root hairs, and comparison with the [163] W. Wei, E. Alexandersson, D. Golldack, A. J. Miller, P. O. closely related potato inwardly rectifying K channel SKT1 Kjellbom, and W. Fricke, “HvPIP1;6, a barley (Hordeum after expression in Xenopus oocytes,” Planta, vol. 210, no. 5, vulgare L.) plasma membrane water channel particularly pp. 723–731, 2000. expressed in growing compared with non-growing leaf [149] J. Xicluna, B. Lacombe, I. Dreyer, et al., “Increased functional tissues,” Plant and Cell Physiology, vol. 48, no. 8, pp. 1132– diversity of plant K channels by preferential heteromer- 1147, 2007. ization of the Shaker-like subunits AKT2 and KAT2,” The [164] W. Lin, Y. Peng, G. Li, et al., “Isolation and functional char- Journal of Biological Chemistry, vol. 282, no. 1, pp. 486–494, acterization of PgTIP1, a hormone-autotrophic cells-specific tonoplast aquaporin in ginseng,” Journal of Experimental [150] B. Sottocornola, S. Visconti, S. Orsi, et al., “The potassium Botany, vol. 58, no. 5, pp. 947–956, 2007. channel KAT1 is activated by plant and animal 14-3-3 [165] W.-G. Choi and D. M. Roberts, “Arabidopsis NIP2;1, a major proteins,” The Journal of Biological Chemistry, vol. 281, no. intrinsic protein transporter of lactic acid induced by anoxic 47, pp. 35735–35741, 2006. stress,” The Journal of Biological Chemistry, vol. 282, no. 33, [151] Q. Leng, R. W. Mercier, B.-G. Hua, H. Fromm, and G. pp. 24209–24218, 2007. A. Berkowitz, “Electrophysiological analysis of cloned cyclic [166] M. Mahdieh, A. Mostajeran, T. Horie, and M. Katsuhara, nucleotide-gated ion channels,” Plant Physiology, vol. 128, “Drought stress alters water relations and expression of PIP- no. 2, pp. 400–410, 2002. type aquaporin genes in Nicotiana tabacum plants,” Plant and [152] B.-G. Hua, R. W. Mercier, Q. Leng, and G. A. Berkowitz, Cell Physiology, vol. 49, no. 5, pp. 801–813, 2008. “Plants do it differently. A new basis for potassium/sodium [167] N. Shitan, I. Bazin, K. Dan, et al., “Involvement of CjMDR1, selectivity in the pore of an ion channel,” Plant Physiology, a plant multidrugresistance-type ATP-binding cassette pro- vol. 132, no. 3, pp. 1353–1361, 2003. tein, in alkaloid transport in Coptis japonica,” Proceedings [153] E. D. Vincill, K. Szczyglowski, and D. M. Roberts, “GmN70 of the National Academy of Sciences of the United States of and LjN70. Anion transporters of the symbiosome mem- America, vol. 100, no. 2, pp. 751–756, 2003. brane of nodules with a transport preference for nitrate,” [168] S. Gustavsson, A.-S. Lebrun, K. Norden, ´ F. Chaumont, Plant Physiology, vol. 137, no. 4, pp. 1435–1444, 2005. and U. Johanson, “A novel plant major intrinsic protein [154] M. A. Piner ˜ os, G. M. A. Canc ¸ ado, and L. V. Kochian, in Physcomitrella patens most similar to bacterial glycerol “Novel properties of the wheat aluminum tolerance organic channels,” Plant Physiology, vol. 139, no. 1, pp. 287–295, acid transporter (TaALMT1) revealed by electrophysiological 2005. characterization in Xenopus oocytes: functional and struc- [169] A.-G. Desbrosses-Fonrouge, K. Voigt, A. Schroder ¨ , S. tural implications,” Plant Physiology, vol. 147, no. 4, pp. Arrivault, S. Thomine, and U. Kramer ¨ , “Arabidopsis thaliana 2131–2146, 2008. MTP1 is a Zn transporter in the vacuolar membrane which [155] A. Meyer, S. Eskandari, S. Grallath, and D. Rentsch, “AtGAT1, mediates Zn detoxification and drives leaf Zn accumulation,” a high affinity transporter for γ-aminobutyric acid in FEBS Letters, vol. 579, no. 19, pp. 4165–4174, 2005. Arabidopsis thaliana,” The Journal of Biological Chemistry, vol. [170] D. Becker, D. Geiger, M. Dunkel, et al., “AtTPK4, an 281, no. 11, pp. 7197–7204, 2006. Arabidopsis tandem-pore K channel, poised to control the 2+ [156] F. Chaumont, F. Barrieu, R. Jung, and M. J. Chrispeels, pollen membrane voltage in a pH- and Ca -dependent “Plasma membrane intrinsic proteins from maize cluster in manner,” Proceedings of the National Academy of Sciences of two sequence subgroups with differential aquaporin activity,” the United States of America, vol. 101, no. 44, pp. 15621– Plant Physiology, vol. 122, no. 4, pp. 1025–1034, 2000. 15626, 2004. [157] C. Dordas, M. J. Chrispeels, and P. H. Brown, “Permeability [171] T. P. Durrett, W. Gassmann, and E. E. Rogers, “The FRD3- and channel-mediated transport of boric acid across mem- mediated efflux of citrate into the root vasculature is brane vesicles isolated from squash roots,” Plant Physiology, necessary for efficient iron translocation,” Plant Physiology, vol. 124, no. 3, pp. 1349–1361, 2000. vol. 144, no. 1, pp. 197–205, 2007. [158] M. Werner, N. Uehlein, P. Proksch, and R. Kaldenhoff, “Characterization of two tomato aquaporins and expression during the incompatible interaction of tomato with the plant International Journal of Peptides Advances in International Journal of BioMed Stem Cells Virolog y Research International International Genomics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Nucleic Acids International Journal of Zoology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com The Scientific Journal of Signal Transduction World Journal Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Advances in Genetics Anatomy Biochemistry Research International Research International Microbiology Research International Bioinformatics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Enzyme Journal of International Journal of Molecular Biology Archaea Research Evolutionary Biology International Marine Biology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Plant Genomics Hindawi Publishing Corporation

Heterelogous Expression of Plant Genes

Loading next page...
 
/lp/hindawi-publishing-corporation/heterelogous-expression-of-plant-genes-IwGoBmzja5
Publisher
Hindawi Publishing Corporation
Copyright
Copyright © 2009 Filiz Yesilirmak and Zehra Sayers. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN
1687-5370
DOI
10.1155/2009/296482
Publisher site
See Article on Publisher Site

Abstract

Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2009, Article ID 296482, 16 pages doi:10.1155/2009/296482 Review Article Filiz Yesilirmak and Zehra Sayers Faculty of Engineering and Natural Sciences, Sabanci University, Orhanli, Tuzla, 34956 Istanbul, Turkey Correspondence should be addressed to Filiz Yesilirmak, filizy@sabanciuniv.edu Received 30 January 2009; Accepted 24 May 2009 Recommended by Pushpendra Gupta Heterologous expression allows the production of plant proteins in an organism which is simpler than the natural source. This technology is widely used for large-scale purification of plant proteins from microorganisms for biochemical and biophysical analyses. Additionally expression in well-defined model organisms provides insights into the functions of proteins in complex pathways. The present review gives an overview of recombinant plant protein production methods using bacteria, yeast, insect cells, and Xenopus laevis oocytes and discusses the advantages of each system for functional studies and protein characterization. Copyright © 2009 F. Yesilirmak and Z. Sayers. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction posttranslational modifications, efficiency of the expression system, as well as simplicity and cost are discussed in the following sections. Heterologous expression involves identification of genes and transfer of the corresponding DNA fragments to hosts other than the original source for synthesis of the encoded 2. Principal Components of proteins. Protein isolation, especially from plant sources, Heterologous Expression can be costly, cumbersome and lengthy, and heterologous expression provides a convenient alternative. This method- Basic principles of heterologous cloning and expression ology allows large-scale production of plant proteins in are summarized in Figure 1. Major parameters that affect microorganisms to study their biochemical and biophysical choices at different stages are also indicated. The choice of features. Foreign hosts may also provide a simpler system for the expression system and vector is a critical step in this studies on functions of proteins and for elucidation of their procedure and, as indicated, advantages and disadvantages roles in complex mechanisms such as metabolic reactions of several factors have to be considered. Expression systems and membrane transport. Recombinant plant proteins and are selected depending on whether the purpose of study is peptides produced by heterologous expression are also used production of large quantities of protein or investigation of in industrial applications. Examples are provided by the functional features of the cloned protein. The physicochem- synthesis of a medicinal peptide from ginseng as potential ical properties of the investigated protein also play a role in drug against diabetes [1] or production of plant lectins [2]in this choice. A general review of frequently used expression both cases in yeast. systems is provided by Yin et al. [3]. The present review covers the recent literature on A comprehensive survey of commercially available plant gene expression in bacteria, yeast, insect cells and expression vectors has recently been published [4]. The Xenopus oocytes and presents the comparative advantages most commonly used vectors are fusion systems that link and disadvantages of each system. It also provides a survey additional amino acid sequences (tags) to the protein of recent examples of application of heterologous expression through a recognition site for a specific protease. Tags may technology to plant proteins. A comprehensive list of plant consist of a short peptide sequence or a full protein which proteins expressed heterologously is given in Table 1.Factors can be cleaved from the protein when desired. Presence influencing the choice of hosts, including the stability of tag sequences facilitates solubility, purification, quan- and folding characteristics of the protein, requirement for tification, identification, localization, and assaying of the 2 International Journal of Plant Genomics Gene(s) •Protein modifications • Solubility • Yield • Fusion partners Host Vector • Cost • Stable expression • Efficiency •Transient expression • Environment/safety/ health Host Vector Expression No Expressed Functional studies Yes (in vivo) Purification •Protein analysis •Structural analysis •Production • Functional studies (in vitro) Figure 1: Flow chart for heterologous expression. expressed protein. Frequently used fusion partners include and relatively low cost. Almost all commercially available glutathione-S-transferease (GST), his-tag (poly-histidines), inducible cloning vectors are compatible with E. coli and maltose binding protein (MBP), thioredoxin (TrxA), FLAG extensive biochemical and genetic information is available. epitope-tag, c-Myc epitope-tag, disulfide isomerase I (DsbA), One of the disadvantages of using E. coli as an expression polyarginine-tag (Arg-tag), calmodulin-binding peptide, host arises from its inability to perform post-translational cellulose-binding domain, poly-histidine affinity tag (HAT- modifications, which are often required for correct fold- tag), N-utilizing substance-A (NusA), S-tag, streptavidin- ing and functional activity of the recombinant protein. binding peptide (SBP-tag), strep-tag, fluorescent proteins This applies particularly to some membrane proteins and (e.g., green fluorescent protein (GFP)) and ubiquitin [4]. enzymes [3]. Another disadvantage is that E. coli is generally MBP and NusA are specifically used to increase the solubility. not suitable for proteins which contain many disulfide MBP is considered to be much more effective for enhancing bonds or require glycosylation, proline cis/trans isomer- solubility than GST and thioredoxin [5]. The major dis- ization, disulfide isomerization, lipidation, sulphation, or advantages of fusion protein systems are the requirement phosphorylation [8]. Some eukaryotic proteins that retain of expensive proteases for cleavage from the recombinant their full biological activity in the nonglycosylated form protein and the low yield of cleavage reactions [6]. have,however,beenproducedin E. coli. The unglycosylated Depending on the host system, vectors for transient or human growth hormone (hGH) binding protein secreted stable expression can be chosen as indicated below. from E.coli retains the same binding affinity and specificity as the wild-type hGH binding protein suggesting that recombinant protein is properly folded and glycosylation is 3. Expression Hosts not required for binding [9]. Production of proteins that are stabilized by disulfide 3.1. Prokaryotic Expression Systems bonds in E.coli often results in proteolytic degradation or 3.1.1. Escherichia Coli. Escherichia coli (E. coli) is the first misfolding and formation of inclusion bodies [6]. One and most extensively used prokaryotic expression system for strategy developed to improve this situation is to target these proteins to the periplasm where the nonreducing heterologous protein production [7]. It remains generally the first choice due to its simplicity, rapid growth rate, environment allows formation of disulfide bonds [10, 11]. In International Journal of Plant Genomics 3 addition, the E.coli periplasm contains chaperone-like disulfide-binding proteins (DsbA, DsbB, DsbC, and DsbD), folding catalysts, and peptidyl-prolyl isomerases (SurA, Cd/protein RotA, FklB, and FkpA) that support disulfide bond forma- GSTdMT 3.4 ± 1 tion and are important for correct folding of periplasmic proteins [12–14]. Disulfide bond formation is achieved via fusion to DsbA or DsbC [15, 16] and periplasmic secretion results in the functional production of a variety of recombinant proteins [17]. In a recent study, the rescue of unstable lipase B from Pseudozyma antarctica (PalB), with periplasmic folding factors was demonstrated [18]. 2 Another strategy involves the use of the trxB gor dou- ble mutant lacking thioredoxin reductase and glutathione reductase genes [19, 20]. This double mutant was used for 220 240 260 280 300 heterologous expression of barley oxalate oxidase (HvOXO) Wavelength (nm) in E.coli [21]. Thegenefor an osmotin-like cryoprotective Figure 2: UV-visible absorption spectrum of GSTdMT at 2.7 protein from Solanum dulcamara was expressed in E.coli mg/mL concentration in 20 mM HEPES buffer at pH 8.0. The and directed to periplasmic localization using an expression charge transfer band between 240 and 260 nm due to Cd-S vector containing the pelB signal sequence [22]. This resulted interaction is indicated by the arrow. The Cd/protein ratio is given in high concentrations of soluble protein with cryoprotective in the inset. activity, whereas expression in the bacterial cytoplasm only yielded large amounts of insoluble and aggregated protein. The seagrass HAK K transporters, CnHAK1 and CnHAK2 Some of the plant proteins accumulated in insoluble were also overexpressed in E.coli and it was found that inclusion bodies in E.coli can be solubilized and refolded CnHAK1, but not CnHAK2, mediated very rapid K or Rb to restore activity after purification from the host. Exam- influxes [35]. Using a dicarboxylate uptake-deficient E.coli ples include Arabidopsis thaumatin-like protein (ATLP3) mutant, a peptide transporter, AgDCAT1 from alder, was which was purified from inclusion bodies and the refolded shown to be a dicarboxylate, including malate, succinate, form displayed activity against some pathogenic fungi [23]. fumarate, and oxaloacetate, transporter [36]. To validate the potential antifungal activity of Solanum E.coli has also been used for expression of small plant nigrum osmotin-like protein (SnOLP) was overexpressed proteins with a fusion partner. Metallothioneins (MTs), in E.coli and the recombinant protein was refolded using which are difficult to purify from natural sources because reduced:oxidized gluthatione redox buffer and its in vitro of their small molecular weight (7 kD), unusual amino acid activity was demonstrated [24]. The soybean RHG1-LRR sequences containing a large number of cysteins and their domain protein was solubilized from inclusion bodies using proteolytic susceptibility belong to this class. Several MTs urea and refolded by removing the urea in the presence of 2+ including a Cd binding Type 1 durum wheat metalloth- arginine and reduced/oxidized glutathione [25]. ionein (dMT) [37], fava bean Type 1 and Type 2 MTs [38], Many plant enzymes are expressed in insoluble inclusion Arabidopsis MT1, MT2 and MT3 proteins [39], Type 3 MT3- bodies but it is still possible to obtain high yields of active A from the oil palm [40], Type 2 MT, QsMT from Quercus forms for structural studies [26]. Thematurepolypeptide of suber [41]havebeenproducedin E.coli mainly for structural FatB thioesterase from the developing seed tissues of Mad- analyses. Since the fusion constructs of durum MT with huca butyracea was characterized by heterologous expression GST (GSTdMT) can be purified in well-defined oligomeric in E.coli [27]. The functionality of the MbFatB in the heterol- states they are used as model systems for studies on metal- ogous system was revealed by the altered growth behavior binding and for structural analyses. Figure 2 illustrates that and cell morphology of the bacteria due to the changes in Cd-binding to GSTdMT can be detected by UV-visible the fatty acid profile. The maize chloroplast transglutaminase spectroscopy. The metal content of GSTdMT was shown to (TGZ) [26] and glutamatecysteine ligase (GCL) [28]were be the same as that expected from dMT alone. An example of efficiently overexpressed in E.coli. Recently, DELLA proteins the shape models generated from X-ray solution scattering from both Arabidopsis and Malus domestica, which are data for GSTdMT is shown in Figure 3, together with the involved in regulation of plant growth in response to phyto- fit to experimental data. The models support a fold for hormonal signals, were isolated and expressed in E. coli [29]. dMT similar to that expected for the free molecule [42, 43]. Examples of functional expression of plant proteins in These results are in agreement with earlier work suggesting E.coli are provided mostly by studies on membrane proteins. independent folding of GST and its fusion components [44] Amutantwithverylow K uptake was used as host and indicate that recombinant fusion complexes are useful as for studies on the K transporters AKT2 [30], AtKUP1- model systems for structural studies. 2[31], AtHKT1 [32] from Arabidopsis and EcHKT1 and EcHKT2 from Eucalyptus camaldulensis [33]. In another example, E.coli C43 strain, which is suitable for expression of 3.2. Eukaryotic Expression Systems. Eukaryotic expression membrane proteins was used for functional characterization systems offer the possibility of posttranslational modifi- of chloroplast ATP/ADP transporter from Arabidopsis [34]. cations and are often used for investigations of protein Absorbance (a.u.) 4 International Journal of Plant Genomics Table 1: Heterologous expression of plant proteins grouped according to the host cells. Protein expressed Plant Reference Escherichia coli Lipase B (PalB) Pseudozyma antarctica [18] Hordeum vulgare, Triticum Oxalate oxidase [21] aestivum Osmotin-like Solanum dulcamara [22] cryoprotective protein Thaumatin-like protein Arabidopsis thaliana [23] (ATLP3) Osmotin-like protein Solanum nigrum [24] (SnOLP) RHG1-LRR domain Glycine max [25] Chloroplast Zea mays [26] transglutaminase (TGZ) FatB thioesterase Madhuca butyracea [27] Glutamatecysteine ligase Arabidopsis thaliana [28] (GCL) Arabidopsis thaliana , Malus DELLA proteins [29] domestica K transporters; KAT1, AKT2-3, AtKUP1/AtKT1/AtPOT1, Arabidopsis thaliana [30–32] AtKUP2/AtKT2/AtPOT2, AtHKT1 K transporters, EcHKT1, Eucalyptus globulus [33] EcHKT2 ATP/ADP transporter Arabidopsis thaliana [34] HAK K transporters, Cymodocea nodosa [35] CnHAK1,CnHAK2 Peptide transporter family Alnus glutinosa [36] member, AgDCAT1 Type 1 MT, dMT Triticum durum [37] Type 1 and Type 2 MTs Vicia faba [38] MT1, MT2, and MT3 Arabidopsis thaliana [39] Type 3 MT3-A Elaeis guineensis [40] Type 2 MT, QsMT Quercus suber [41] Soybean seed ferritin Glycine max [126] Saccharomyces cerevisiae H -amino acid symporter Arabidopsis thaliana [47] and K channel, KATl Phosphate transporters; Arabidopsis thaliana [48] AtPT1 and AtPT2 K transporter, HvHAKI Hordeum vulgare [49] K transporters, AtKT1, Arabidopsis thaliana [50, 51] and AtKT2, AtKUP1 K transporter, HKT1 Triticum aestivum [52, 53] Sulfate transporters, Lycopersicon esculentum [54] LeST1-1 and LeST1-2 Copper transporters, Arabidopsis thaliana [55] (COPT1–5) Peptide transporter, Arabidopsis thaliana [56] AtPTR1 + + K /H antiporter, AtChx17 Arabidopsis thaliana [57] International Journal of Plant Genomics 5 Table 1: Continued. Protein expressed Plant Reference Hexose transporters, Vitis vinifera [58] VvHT4 and VvHT5 Plasma membrane-localized Arabidopsis thaliana [59] H /inositol symporter, AtINT2 High affinity Arabidopsis thaliana [60] GABAtransporter, AtGAT1 Tonoplast Intrinsic Proteins, AtTIP2;1 and Arabidopsis thaliana [61] AtTIP2;3 Sorbitol transporters, Plantago major [62] PmPLT1 and PmPLT2 Pichia pastoris Nitrate reductase Spinacia oleracea,Zea mays [69] Invertase Ipomoea batatas [70] α1,6-galactosyltransferase Trigonella foenum-graecum [71] α1,6-xylosyltransferase Arabidopsis thaliana [72] Arabidopsis thaliana Bos taurus, Drosophila Glycosyltransferases melanogaster, [73] Caenorhabditis elegans, Leucopersicon esculentum β-D-fructofuranosidase Oryza sativa [74] Apyrase Solanum tuberosum [75] Oxalate oxidases, HvOXO, Hordeum vulgare, Triticum [76, 77] TaOXO aestivum Canavalia brasiliensis, Lectin [2, 79] Nicotiana tabacum Low-affinity cation Triticum aestivum [80] transporter (LCT1) 2S albumin storage Glycine max [127] proteins (AL1 and AL3) Baculovirus-mediated insect cell Patatin Solanum tuberosum [81] Reductase isoforms, AR1 Arabidopsis thaliana [82] and AR2 Peroxisomal short-chain Arabidopsis thaliana [83] acyl-CoA oxidase A Cyclin-dependent kinase A Arabidopsis thaliana [84] (CDKA) NADH-cytochrome (Cyt) Arabidopsis thaliana [85] b5 reductase Geranylgeranyltransferase-I Arabidopsis thaliana [86] (GGT-I) Acyl-CoA synthetase Arabidopsis thaliana [87] Homogentisate Arabidopsis thaliana [88] phytyltransferase (+)-Abscisic Acid Arabidopsis thaliana [89] 8’-Hydroxylase β1,2-xylosyltransferase Arabidopsis thaliana [90] Ethylene-inducing xylanase Nicotiana tabacum [91] 6 International Journal of Plant Genomics Table 1: Continued. Protein expressed Plant Reference ADP-glucose pyrophoshorylase Hordeum vulgare [92] (AGPase) K channels, AKT1, KAT1, Arabidopsis thaliana [93–95] and KCO1 K channels KST1, SKT1, Solanum tuberosum [96, 97] and KST1 Transporter AUX1 Arabidopsis thaliana [98] β-phaseolin polypeptides Phaseolus vulgaris [128] Ac-specific ORFa protein, Zea mays [129] Cysteine protease papain Carica papaya [130] Mitochondrial protein Zea mays [131] URF13 LAT52 protein Lycopersicon esculentum [132] Auxin-binding protein Zea mays, Nicotiana [133, 134] (ABP1) tabacum Calreticulin and auxin Zea mays [135] binding protein Cinnamate 4-Hydroxylase Arabidopsis thaliana [136] Cryptochrome-1 Arabidopsis thaliana [137] Phototropin 2 Arabidopsis thaliana [138] Histidinol dehydrogenase Brassica oleracea [139] Putative soluble epoxide Solanum tuberosum [140] hydrolase (sEH) lmidazoleglycerolphosphate Arabidopsis thaliana [141] dehydratase Phytone synthase, Narcissus pseudonarcissus [142, 143] Phytoene desaturase 4-coumarate:coenzyme A Populus trichocarpa, [144] ligase (4Cl) Populusdeltoides Xenopuslaevisoocytes Na − K cotransporter Arabidopsis thaliana [39] HKT1 AgDCAT1 nodule-specific Alnus glutinosa [43] transporter AtNAR2.1/AtNRT2 Nitrate Arabidopsis thaliana [102] Transport System HKT Constructs, Triticum aestivum, [103] AtHKT1 HKT1 chimeras Arabidopsis thaliana HKT1 superfamily of Eucalyptus camaldulensis [104] K /Na transporters Ammonium transporter, Lycopersicon esculentum [105] LeAMT1 Ammonium transporter, Arabidopsis thaliana [106] AtAMT1;2 Sucrose transporters, Arabidopsis thaliana,Lotus [107–109] AtSUC2, AtSUC9, LjSUT4 japonicus Al-activated malate transporter, Brassica napus, Triticum [110, 111] BnALMT1,BnALMT2, aestivum ALMT1 Polyol transporters, Arabidopsis thaliana, [112, 113] AtPLT5, PmPLT1 Plantago major International Journal of Plant Genomics 7 Table 1: Continued. Protein expressed Plant Reference Inositol transporter2, Arabidopsis thaliana [114, 115] AtINT2, AtINT4 Amino acid transporter, Arabidopsis thaliana [116] AtCAT6, Cation–Cl- cotransporter, Arabidopsis thaliana [117] CCC Anion-selective Zea mays [118] transporter, ZmALMT1 K channel, SIRK Vitis vinifera [145] K channel, KZM1 Zea mays [146] K channel, ZMK1 Zea mays [147] K channels, SKT1 and Solanum tuberesum, [148] LKT1 Lycopersicon esculentum AKT2-KAT2 subunitits Arabidopsis thaliana [149] K channel, KAT1 Arabidopsis thaliana [150] Cyclic nucleotide-gated ion Arabidopsis thaliana, channels AtCNGC2, [151, 152] Nicotiana tobacum AtCNGC1, -2 Putative transporter Glycine max [153] (GmN70) Al-activated malate Triticum aestivum [154] transporter, TaALMT1 High affinity γ-aminobutyric acid Arabidopsis thaliana [155] transporter, AtGAT1 Aquaporins, ZmPIP1a, ZmPIP1b, ZmPIP2a, PIP1, Zea mays [124, 156, 157] ZmPIP2;1 Aquaporin, PIP1 Lycopersicon esculentum [158] Aquaporin, PIP2 Juglans regia [159] Tonoplast intrinsic protein, Arabidopsis thaliana [160, 161] AtTIP2;1 Mesembryanthemum Aquaporin, McTIP1;2 [162] crystallinum Aquaporin, HvPIP1;6 Hordeum vulgare [163] Tonoplast intrinsic protein, Panax ginseng [164] PgTIP1 Nodulin 26 intrinsic Arabidopsis thaliana [165] protein, AtNIP2;1 PIP-1-type; NtPIP1;1, NtAQP1; PIP-2-type; Nicotiana tabacum [166] NtPIP2;1 CjMDR1, ATP-binding Coptis japonica [167] cassette protein GlpF-like intrinsic protein Physcomitrella patens [168] (GIP1;1), Metal tolerance protein1, Arabidopsis thaliana [169] AtMTP1 AtTPK4 tandem-pore Arabidopsis thaliana [170] K channel FRD3, multidrug and toxin Arabidopsis thaliana [171] efflux (MATE) 8 International Journal of Plant Genomics −1 −2 −3 0 0.1 0.2 0.3 −1 S (nm ) (a) (b) Figure 3: A: a low resolution shape model for GSTdMT. The GST dimer (red and blue) is located at the center from which the dMT molecules extend (green). B: the scattering curve expected from the model (−) agrees well with the experimental data (...). I(S) is the scattering intensity and S the scattering vector given by S = 4π sinθ/λ, where 2θ is the scattering angle and λ= 1.5 A is the wavelength of X-rays. The model and the expected scattering pattern were calculated using the programs in the ATSAS package (EMBL Hamburg Outstation). function. Processing reactions such as O-and N -linked gly- bidopsis [51] also utilized S. cerevisiae mutants. Another K cosylation, tyrosine, serine, and threonine phosphorylation, transporter characterized in this system is HKT1 from wheat addition of fatty acid chains, processing of signal sequences, [52, 53]. Kinetic uptake analyses of tomato sulfate trans- disulfide bond formation, and correct folding can all be porters, LeST1-1 and LeST1-2 were carried out using the S. readily performed in eukaryotic hosts. The most commonly cerevisiae sulfate transporter mutant [54]. The five members used eukaryotic systems are yeast, insect, mammalian, and of the copper transporter family COPT1–5 from Arabidopsis plant cells. were characterized using a copper transport null mutant [55]. A peptide transporter AtPTR1 gene from Arabidopsis 3.2.1. Yeast. As a single cell eukaryotic organism, yeast has was isolated and complemented in a peptide transport- + + deficient mutant [56]. A putative K /H antiporter, AtChx17 molecular, genetic, and biochemical characteristics which are similar to those of higher eukaryotes, and is useful for was heterologously expressed and characterized in an S. heterelogous protein production. Yeast cells can grow rapidly cerevisiae kha1 deletion mutant [57]. To test their functional activity, the grapevine hexose transporters VvHT3, VvHT4, with high cell densities, and are easy to manipulate and yeast cultures are cost effective. The two most commonly used and VvHT5 were expressed in the S. cerevisiae mutant EBY.VW4000, which is deficient in glucose transport due organisms are Saccharomyces cerevisiae (S. cerevisiae)and Pichia pastoris (P. pastoris) [7]. to concurrent knock-out of 20 endogenous transporter genes [58]. Growth-based complementation assays were used to demonstrate function of the transporters but resulted Saccharomyces Cerevisiae. Baker’s yeast, S. cerevisiae,is widely used as a host organism for heterologous expression in inadequate rates of glucose uptake. A more sensitive assay based on direct measurement of radioactively labelled of proteins. Its genetics and physiology are well documented glucose uptake revealed that this mutant expressing VvHT4 and proteins are posttranslationally modified through the and VvHT5 accumulated labelled glucose at higher rates than mechanisms similar to those found in plants. The limitations yeast transformed with the empty vector, demonstrating of this host system are low yields, cell stress due to the pres- the functionality of the glucose transporters. Although ence of the foreign gene and hyperglycosylation of secreted VvHT3:GFP (green fluorescent protein) fusion protein was foreign proteins. Lack of a strong inducible promoter can be targeted to the plasma membrane in plant cells, VvHT3 was circumvented using P. pastoris [45]. Earlier work on heterelogous expression for screening of found not to be functional in the yeast system [58].Yeast expression studies were, in several instances, complemented plant cDNA libraries by complementation in S. cerevisiae by studies in other organisms to verify functional and kinetic null mutants was reviewed by Frommer and Ninnemann [7]. The S. cerevisiae mutants provide a convenient system properties of recombinant proteins. The plasma membrane- localized H /inositol symporter AtINT2 of Arabidopsis was for functional and kinetic studies of transporters [46]. The studied by expression in an inositol uptake/inositol biosyn- electrophysiological properties of membrane transporters, + + thesis double mutant in S. cerevisiae and in Xenopus oocytes H -amino acid symporter and K channel, KAT1 [47]and [59]. In this study, the amount of AtINT2 protein in yeast phosphate transporters; AtPT1 and AtPT2 of Arabidopsis were characterized using S. cerevisiae [48]. Recently, func- plasma membrane was sufficient for complementation, but not for functional and kinetic analyses. In oocytes, however, tional expression of transporters such as an HvHAKI from it was possible to show that AtINT2 mediated the symport barley [49], AtKT1 and AtKT2 [50], and AtKUP1 from Ara- log I (S) International Journal of Plant Genomics 9 of H [59]. Expression and functional characterization of [73]. To confirm that Osβfruct3 from rice encoded a vac- Arabidopsis AtGAT1 in S. cerevisiae and Xenopus oocytes uolar type β-D-fructofuranosidase, the Osβfruct3 cDNA was revealed that AtGAT1 mediates H -dependent, high affinity expressed in this host [74]. A recombinant potato apyrase transport of high affinity γ-aminobutyric acid (GABA) was expressed and purified in the hyperglycosylated form at and GABA-related compounds. Properties of this protein 1 mg/L protein concentration [75]. The catalytically active couldbeexaminedinmoredetailin Xenopus oocytes [60]. barley oxalate oxidase, HvOXO was produced with a yield of Heterologous expression of AtTIP2;1 and AtTIP2;3 from 50 mg/L culture and biochemically characterized [76]. Arabidopsis in both ammonium uptake-defective yeast and High-level expression of wheat germin/oxalate oxidase oocytes indicated that these TIPs transport both ammonium was achieved in P. pastorisas an α-mating factor signal pep- and methyl-ammonium in addition to water and urea tide fusion to increase secretion of the protein of interest into [61]. The kinetic characteristics of the sorbitol transporters, the culture medium. Approximately 1 g (4×10 U) of TaOXO PmPLT1, and PmPLT2 from common plantain (Plantago was produced in 5 L fermentation cultures following 8 days of major) were investigated by functional expression in S. methanol induction, demonstrating the possibility of large- cerevisiae and in Xenopus oocytess. In the yeast system, scale production of oxalate oxidase for biotechnological both proteins were characterized as low-affinity and low- applications. Glycosylation of the recombinant protein was specificity polyol symporters. These data were confirmed in evidencedbymassspectrometry[77]. Another application the Xenopus system, where PmPLT1 was analyzed in detail using P. pastoris is the expression of the α-subunit of and characterized as an H symporter [62]. heterotrimeric G-proteins, GPA1, from Arabidopsis. Several The major disadvantages of using S. cerevisiae mutants attempts had previously failed to produce this protein in in transporter studies are the hyperpolarization of the mem- E. coli, whereas in the yeast system the protein could be brane, mislocalization of membrane proteins and recruit- expressed with a his -tag and purified by affinity chromatog- + + ment of non-K -transporters into K -transporters [63]. raphy with a yield up to 20 mg from 700 mL culture [78]. Several allergens including, Cyn d 1 from Bermuda grass, Pichia Pastoris. P. pastoris, methylotrophic yeast, is consid- Bla g 4 from German cockroach,Amb a6from Ambrosia artemisiifolia, andOlee1from Olea europaea have also been ered a valuable tool for high yield heterologous expression of various proteins. The possibility of obtaining posttransla- produced in P. pastoris (see list in 64). tional modifications, high level expression of foreign proteins This system was also used for the expression of a number of plant lectins such as Canavalia brasiliensis lectin (ConBr) in either intracellular or extracellular forms, simplicity of genetic manipulations, and availability of various P. [2] and the Nicotiana tabacum lectin [79]. In a recent pastoris strains and vectors make this expression system study, the low-affinity cation transporter (LCT1) from wheat highly popular [64]. Molecular manipulations such as gene was also expressed and functionally characterized using P. targeting, high frequency DNA transformation, and cloning pastoris [80]. for functional complementation are similar to those in S. cerevisiae [64]. Tightly regulated promoters, easy integration 3.2.2. Insect Cells. Baculoviruses have been used for the of heterologous DNA into the host chromosome and the synthesis of a wide variety of eukaryotic recombinant capacity to generate more posttranslational modifications proteins in insect cells. In this expression system one of make P. pastoris the preferred system compared to S. the nonessential viral genes is replaced with the target cerevisiae. protein through homologous recombination. The resulting The wide use of P. pastoris expression system for reco- recombinant baculovirus is used to infect cultured insect mbinant plant proteins can be seen from recent reviews cells and the heterologous genes can be expressed under the [64, 65]. P. pastoris is particularly well suited for studying control of the extremely strong pPolh, polyhedron promoter plant enzymes since glycosylation of the foreign proteins in the late phase of infection. is expected to be closer to that in plants [66, 67]and The most common baculovirus used for expression glycosylated proteins have shorter glycosyl chains in P. studies is Autographa californica multiple capsid nucle- pastoris than in S. cerevisiae [68]. This expression system has opolyhedrovirus (AcMNPV) and the most frequently used the potential to produce high levels of recombinant proteins host insects are Spodoptera frugiperda and Trichoplusia ni. [67], up to 400 mg/L of culture [69]. Several plant enzymes This expression system produces high levels of recombinant have been produced in Pichia. Two examples are cytosolic proteins which are soluble, post-translationally modified, expression of nitrate reductase from spinach and corn at biologically active, and functional [81]. The virus is not high levels needed for detailed biochemical studies [69]and pathogenic to vertebrates or plants. The main drawback of expression of a sweet potato invertase in milligram quantities this system over the bacterial and yeast systems lies in the [70]. Enzymatic activity of the membrane-bound α1,6- noncontinuous expression of the heterologous gene; every galactosyltransferase was shown through overexpression in round of protein production needs reinfection [3]. P. pastoris [71]. The hypothesis that α-xylosyltransferase is This heterologous expression system is mainly used to involved in xyloglucan biosynthesis was tested by overex- investigate enzymatic mechanisms in plants. The most recent pressing the corresponding genes and identifying the gene examples include the Arabidopsis reductase isoforms, AR1 product that displayed activity [72]. P. pastoris has been used and AR2 [82], peroxisomal short-chain acyl-CoA oxidase A for production of a number of glycosyltransferases involved [83], cyclin-dependent kinase A [84], NADH-cytochrome in the biosynthesis of N-and O-linked oligosaccharides b5 reductase [85], geranylgeranyltransferase-I [86], acyl-CoA 10 International Journal of Plant Genomics synthetase [87], homogentisate phytyltransferase [88], (+)- transporters [110, 111], polyol transporters [112, 113], inos- abscisic acid 8’-hydroxylase [89], β1,2-xylosyltransferase itol transporters [114, 115], an amino acid transporter [116], [90], tobacco ethylene-inducing xylanase [91], and barley a cation–Cl-cotransporter [117], and an anion-selective ADP-glucose pyrophoshorylase [92]. The overall yield of transporter [118]in Xenopus oocytes were investigated. heterelogous proteins obtained with this system is usually Cases where channel proteins expressed in oocytes were not lower than with P. pastoris. functional have also been reported. These include the K Baculovirus-infected insect cells have been used as an channels AKT1 from Arabidopsis [93, 119, 120], TaAKT1 alternative system to Xenopus oocytes for expression and from wheat [121], DKT1 from carrot [122], and OsAKT1 characterization of plant channel proteins. Several channel from rice [123]. The causes for the lack of function of these proteins which were not functional in oocytes could be recombinant proteins are not clear. characterized in baculovirus-infected insect cells such as the Several studies have used expression of a wild type and K channel proteins AKT1 [93], KAT1 [94], KCO1 [95]from its mutant forms in Xenopus oocytes to confirm the in Arabidopsis, and KST1 [96]and SKT1 [97]frompotato. vivo functions of plant proteins, especially transporters and To investigate the interaction between AUX1 and its plasma membrane intrinsic proteins (PIPs or aquaporins). transport substrate indole-3-acetic acid (IAA) from Ara- To demonstrate whether or not the plant K channels form bidopsis, an epitope-tagged version of AUX1 was expressed multimers, the wild type and a mutant were coexpressed in at high levels in a baculovirus expression system and suitable Xenopus oocytes [120]. Coexpression of tomato ammonium membrane fragments were prepared from baculovirus- transporter (LeAMT1;1) and its mutant in Xenopus oocytes infected insect cells for direct measurement of IAA binding inhibited ammonium transport, suggesting homooligomer- to AUX1. AUX1-IAA interactions were determined using a ization [105]. In another study, the role of phosphorylation radio-ligand binding assay to confirm that AUX1 was able to in the water channel activity of wild-type and mutant bind IAA with an affinity (Kd) of 2.6 mM, comparable with ZmPIP2;1 was studied in Xenopus oocytes [124]. estimates of the Km for IAA transport [98]. In recent studies, the Xenopus oocyte expression system The main disadvantages of using baculovirus-infected was used to investigate structure-function relationships. In insect cells are difficulties in constructing the expression one example, differences in the function of two cation vectors, requirements for more complex laboratory facilities transporters, wheat HKT1 and Arabidopsis AtHKT1, were and skills, and the short expression periods after infection. investigated using a series of AtHKT1/HKT1 chimeras with point mutations [103]. 3.2.3. Xenopus Laevis Oocytes. The oocytes of the South African clawed frog, Xenopus laevis, are also used for 4. Conclusions heterelogous expression of eukaryotic genes. The mRNA for the target protein, introduced by microinjection into the Heterologous expression of plant genes in other host cytoplasm, is translated and the protein is posttranslationally organisms has two main applications: (1) overexpression modified by the oocyte [99]. Direct injections of DNA into of the encoded protein, for biochemical and biophysical the nucleus are also possible, but the manipulations are characterization and (2) expression of foreign genes for difficult as the nucleus can easily be damaged in the process. determination of the function of the encoded protein Investigations on membrane transport proteins can be by complementing in a mutant host. Overexpression of readily performed on oocytes where techniques for electro- recombinant proteins is usually carried out with a cleavable physiological measurements are well established. Although, tag to simplify purification in large quantities. In contrast, a high proportion of cells express the foreign gene after complementation studies are carried out in null mutants to injection variations in the quality of oocytes and in the ability restore a missing activity in vivo. of individual cells to produce the heterelogous protein can Decisions on which expression vectors to use and the cause problems. Oocytes are not suitable for preparing large choice of the expression host depend on the particular application. In general E.coli is the first choice as host because quantities of proteins and the short expression period often leads to technical difficulties. The system can also not be of its simplicity, availability of expression vectors, cost effec- sustained over long periods of time and is not suitable for tiveness, and availability of extensive genetic information on stable expression [99, 100]. this host. Alternative expression systems are used only if Xenopus oocytes have,however,providedapowerful the recombinant protein is inactive due to lack of essential heterologous expression system for animal as well as plant posttranslational modifications and when detailed studies genes.The possibility of using Xenopus oocytes as heterol- on the recombinant protein function are planned. Yeast ogous expression systems for the identification of plant systems have the advantage of ease of manipulation and short transporters was first demonstrated by the expression of generation time. S. cerevisiae has been extensively used for the H /glucose transporter STP1 from Arabidopsis [101]. It functional complementation, biochemical, and electrophysi- has, since, been mainly used for production of transporters olagical characterization of plant membrane and transporter including potassium channels, H /hexose cotransporters, proteins. P. pastoris is the preferred host for overexpression aquaporins, and chloride channels [99]. In addition, func- of several plant enzymes. Baculovirus-mediated insect cell tional expression of a nitrate transporter [102],aK /Na expression offers the possibility for detailed investigations of transporter [39, 103, 104], ammonium transporters [105, plant enzymes and transporters. The oocyte from Xenopus 106], sucrose transporters [107–109], Al-activated malate laevis is often used for monitoring activity and biochemical International Journal of Plant Genomics 11 and electrophysiological characterization of plant plasma prospects,” Current Opinion in Biotechnology, vol. 16, no. 5, pp. 538–545, 2005. membrane transporter and pump proteins. [12] A. Shokri, A. M. Sanden, ´ and G. Larsson, “Cell and Heterologous expression is a powerful tool for functional process design for targeting of recombinant protein into the and biochemical analyses of genes and gene families isolated culture medium of Escherichia coli,” Applied Microbiology and from various organisms. It is particularly important for Biotechnology, vol. 60, no. 6, pp. 654–664, 2003. plants where the whole genome sequence is not available. [13] F. Baneyx and M. Mujacic, “Recombinant protein folding and This system will also provide denovo analysis. Its limita- misfolding in Escherichia coli,” Nature Biotechnology, vol. 22, tions, however, should be kept in mind, especially when no. 11, pp. 1399–1408, 2004. interpreting the results in terms of the native structure and [14] J. H. Choi and S. Y. Lee, “Secretory and extracellular function of proteins. Major problems arise from misfolding production of recombinant proteins using Escherichia coli,” and mislocalization of recombinant proteins in foreign hosts. Applied Microbiology and Biotechnology,vol. 64, no.5,pp. Strategies developed to avoid misfolding of recombinant 625–635, 2004. [15] Y. Kurokawa, H. Yanagi, and T. Yura, “Overproduction of proteins include expression in periplasmic space, expression bacterial protein disulfide isomerase (DsbC) and its modu- with a tag, and utilization of different hosts. Mislocalization, lator (DsbD) markedly enhances periplasmic production of on the other hand, may occur because the recombinant human nerve growth factor in Escherichia coli,” The Journal of protein may take over the function of the missing host Biological Chemistry, vol. 276, no. 17, pp. 14393–14399, 2001. protein [125]. Conclusions on function need to be tested in [16] S. Sahdev, S. K. Khattar, and K. S. Saini, “Production of active alternative hosts and eventually in the plant itself. eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies,” Molecular References and Cellular Biochemistry, vol. 307, no. 1-2, pp. 249–264, [1] Y. Yan, J. Chen, and J. Li, “Overexpression of a small 2008. [17] F. J. M. Mergulha ˜o,D.K.Summers,and G. A. Monteiro, medicinal peptide from ginseng in the yeast Pichia pastoris,” Protein Expression and Purification, vol. 29, no. 2, pp. 161– “Recombinant protein secretion in Escherichia coli,” Biotech- 166, 2003. nology Advances, vol. 23, no. 3, pp. 177–202, 2005. [18] Y. Xu, D. Lewis, and C. P. Chou, “Effect of folding factors [2] W. M. Bezerra, C. P. Carvalho, R. A. Moreira, and T. B. Grangeiro, “Establishment of a heterologous system for the in rescuing unstable heterologous lipase B to enhance its overexpression in the periplasm of Escherichia coli,” Applied expression of Canavalia brasiliensis lectin: a model for the study of protein splicing,” Genetics and Molecular Research, Microbiology and Biotechnology, vol. 79, no. 6, pp. 1035–1044, vol. 5, no. 1, pp. 216–223, 2006. [3] J. Yin, G. Li, X. Ren, and G. Herrler, “Select what you need: a [19] P. H. Bessette, F. Aslund, J. Beckwith, and G. Georgiou, “Efficient folding of proteins with multiple disulfide bonds comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes,” Journal in the Escherichia coli cytoplasm,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, of Biotechnology, vol. 127, no. 3, pp. 335–347, 2007. [4] K. Terpe, “Overview of tag protein fusions: from molecular no. 24, pp. 13703–13708, 1999. [20] W. A. Prinz, F. Aslund, A. Holmgren, and J. Beckwith, and biochemical fundamentals to commercial systems,” Applied Microbiology and Biotechnology,vol. 60, no.5,pp. “The role of the thioredoxin and glutaredoxin pathways 523–533, 2003. in reducing protein disulfide bonds in the Escherichia coli cytoplasm,” The Journal of Biological Chemistry, vol. 272, no. [5] R. B. Kapust and D. S. Waugh, “Escherichia coli maltose- binding protein is uncommonly effective at promoting the 25, pp. 15661–15667, 1997. [21] P. Cassland, S. Larsson, N.-O. Nilvebrant, and L. J. Jonsson, ¨ solubility of polypeptides to which it is fused,” Protein Science, vol. 8, no. 8, pp. 1668–1674, 1999. “Heterologous expression of barley and wheat oxalate oxi- dase in an E. coli trxB gor double mutant,” Journal of [6] F. Baneyx, “Recombinant protein expression in Escherichia coli,” Current Opinion in Biotechnology,vol. 10, no.5,pp. Biotechnology, vol. 109, no. 1-2, pp. 53–62, 2004. [22] S. S. Newton andJ.G.Duman,“An osmotin-like cryopro- 411–421, 1999. [7] W. B. Frommer and O. Ninnemann, “Heterologous expres- tective protein from the bittersweet nightshade Solanum dulcamara,” Plant Molecular Biology, vol. 44, no. 5, pp. 581– sion of genes in bacterial, fungal, animal, and plant cells,” Annual Review of Plant Physiology and Plant Molecular 589, 2000. [23] X. Hu and A. S. N. Reddy, “Cloning and expression of a PR5- Biology, vol. 46, pp. 419–444, 1995. [8] A.Lueking,C.Holz,C.Gotthold,H.Lehrach,and D. Cahill, like protein from Arabidopsis: inhibition of fungal growth by “A system for dual protein expression in Pichia pastoris and bacterially expressed protein,” Plant Molecular Biology, vol. 34, no. 6, pp. 949–959, 1997. Escherichia coli,” Protein Expression and Purification, vol. 20, no. 3, pp. 372–378, 2000. [24] M. de A Campos, M. S. Silva, C. P. Magalhaes, ˜ et al., “Expression in Escherichia coli, purification, refolding and [9] G. Fuh, M. G. Mulkerrin, S. Bass, et al., “The human growth hormone receptor. Secretion from Escherichia coli antifungal activity of an osmotin from Solanum nigrum,” Microbial Cell Factories, vol. 7, pp. 7–17, 2008. and disulfide bonding pattern of the extracellular binding domain,” The Journal of Biological Chemistry, vol. 265, no. 6, [25] A. J. Afzal and D. A. Lightfoot, “Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded pp. 3111–3115, 1990. [10] C. Wulfing ¨ and A. Pluc ¨ kthun, “Protein folding in the from Escherichia coli inclusion bodies: preparation for a functional analysis,” Protein Expression and Purification, vol. periplasm of Escherichia coli,” Molecular Microbiology, vol. 12, no. 5, pp. 685–692, 1994. 53, no. 2, pp. 346–355, 2007. [26] P. K. Carvajal-Vallejos, A. Campos, P. Fuentes-Prior, et al., [11] G. Georgiou and L. Segatori, “Preparative expression of secreted proteins in bacteria: status report and future “Purification and in vitro refolding of maize chloroplast 12 International Journal of Plant Genomics transglutaminase over-expressed in Escherichia coli,” Biotech- [41] G. Mir, J. Domenec ` h, G. Huguet, et al., “A plant type 2 nology Letters, vol. 29, no. 8, pp. 1255–1262, 2007. metallothionein (MT) from cork tissue responds to oxidative [27] J. K. Jha, M. K. Maiti, A. Bhattacharjee, A. Basu, P. C. Sen, stress,” Journal of Experimental Botany, vol. 55, no. 408, pp. and S. K. Sen, “Cloning and functional expression of an acyl- 2483–2493, 2004. ACP thioesterase FatB type from Diploknema (Madhuca) [42] F. Dede,G.Dinler,and Z. Sayers,“3D Macromolecular butyracea seeds in Escherichia coli,” Plant Physiology and structure analyses: applications in plant proteins,” in Proceed- Biochemistry, vol. 44, no. 11-12, pp. 645–655, 2006. ings of the NATO Advanced Research Workshop, pp. 135–146, [28] J. M. Jez, R. E. Cahoon, and S. Chen, “Arabidopsis Springer, 2006. thaliana glutamate-cysteine ligase. Functional properties, [43] F. Yesilirmak, Biophysical and functional characterization of kinetic mechanism, and regulation of activity,” The Journal of wheat metallothionein at molecular level, Ph.D. thesis, Sabanci Biological Chemistry, vol. 279, no. 32, pp. 33463–33470, 2004. University, Istanbul, Turkey, 2008. [29] X. Sun, N. Frearson, C. Kirk, et al., “An E. coli expression [44] Y. Zhan, X. Song, and G. W. Zhou, “Structural analysis system optimized for DELLA proteins,” Protein Expression of regulatory protein domains using GST-fusion proteins,” and Purification, vol. 58, no. 1, pp. 168–174, 2008. Gene, vol. 281, no. 1-2, pp. 1–9, 2001. [30] N. Uozumi, T. Nakamura, J. I. Schroeder, and S. Muto, [45] M. Schmidt and D. R. Hoffman, “Expression systems for “Determination of transmembrane topology of an inward- production of recombinant allergens,” International Archives rectifying potassium channel from Arabidopsis thaliana based of Allergy and Immunology, vol. 128, no. 4, pp. 264–270, 2002. on functional expression in Escherichia coli,” Proceedings of [46] I. Dreyer, C. Horeau, G. Lemaillet, et al., “Identification the National Academy of Sciences of the United States of and characterization of plant transporters using heterologous America, vol. 95, no. 17, pp. 9773–9778, 1998. expression systems,” Journal of Experimental Botany, vol. 50, [31] E. J. Kim, J. M. Kwak,N.Uozumi, andJ.I.Schroeder, pp. 1073–1087, 1999. “AtKUP1:an Arabidopsis gene encoding high-affinity potas- [47] A. Bertl, J. A. Anderson, C. L. Slayman, and R. F. Gaber, sium transport activity,” The Plant Cell, vol. 10, no. 1, pp. 51– “Use of Saccharomyces cerevisiae for patch-clamp analysis 62, 1998. of heterologous membrane proteins: characterization of [32] N. Uozumi, E. J. Kim, F. Rubio, et al., “The Arabidopsis HKT1 Kat1, an inward-rectifying K channel from Arabidopsis gene homolog mediates inward Na currents in Xenopus thaliana, and comparison with endogeneous yeast channels laevis oocytes and Na uptake in Saccharomyces cerevisiae,” and carriers,” Proceedings of the National Academy of Sciences Plant Physiology, vol. 122, no. 4, pp. 1249–1259, 2000. of the United States of America, vol. 92, no. 7, pp. 2701–2705, [33] D. J. Fairbairn, W. Liu, D. P. Schachtman, S. Gomez-Gallego, 1995. S. R. Day, and R. D. Teasdale, “Characterisation of two [48] U. S. Muchhal, J. M. Pardo, and K. G. Raghothama, distinct HKT1-like potassium transporters from Eucalyptus “Phosphate transporters from the higher plant Arabidopsis camaldulensis,” Plant Molecular Biology, vol. 43, no. 4, pp. thaliana,” Proceedings of the National Academy of Sciences of 515–525, 2000. the United States of America, vol. 93, no. 19, pp. 10519–10523, [34] J. Tjaden, C. Schwopp ¨ e, T. Mohlmann, ¨ P. W. Quick, and 1996. H. E. Neuhaus, “Expression of a plastidic ATP/ADP trans- [49] G. E. Santa-Mar´ ıa, F. Rubio, J. Dubcovsky, and A. Rodr´ ıguez- porter gene in Escherichia coli leads to a functional adenine Navarro, “The HAK1 gene of barley is a member of a nucleotide transport system in the bacterial cytoplasmic large gene family and encodes a high-affinity potassium membrane,” The Journal of Biological Chemistry, vol. 273, no. transporter,” The Plant Cell, vol. 9, no. 12, pp. 2281–2289, 16, pp. 9630–9636, 1998. 1997. [35] B. Garciadeblas, B. Benito, and A. Rodr´ ıguez-Navarro, [50] F. J. Quintero and M. R. Blatt, “A new family of K “Molecular cloning and functional expression in bacteria of transporters from Arabidopsis that are conserved across the potassium transporters CnHAK1 and CnHAK2 of the phyla,” FEBS Letters, vol. 415, no. 2, pp. 206–211, 1997. seagrass Cymodocea nodosa,” Plant Molecular Biology, vol. 50, [51] H.-H. Fu and S. Luan, “AtKUP1: a dual-affinity K trans- no. 4-5, pp. 623–633, 2002. porter from arabidopsis,” The Plant Cell,vol. 10, no.1,pp. [36] J. Jeong, S. Suh, C. Guan, et al., “A nodule-specific dicar- 63–73, 1998. boxylate transporter from alder is a member of the peptide [52] D. P. Schachtman and J. I. Schroeder, “Structure and transporter family,” Plant Physiology, vol. 134, no. 3, pp. 969– transport mechanism of a high-affinity potassium uptake 978, 2004. transporter from higher plants,” Nature, vol. 370, no. 6491, [37] K. Bilecen, U. H. Ozturk, A. D. Duru, et al., “Triticum pp. 655–658, 1994. durum metallothionein: isolation of the gene and structural [53] F. Rubio, W. Gassmann, and J. I. Schroeder, “Sodium-driven characterization of the protein using solution scattering and potassium uptake by the plant potassium transporter HKT1 molecular modeling,” The Journal of Biological Chemistry, and mutations conferring salt tolerance,” Science, vol. 270, vol. 280, no. 14, pp. 13701–13711, 2005. no. 5242, pp. 1660–1663, 1995. [38] R. C. Foley, Z. M. Liang, and K. B. Singh, “Analysis of type [54] J. R. Howarth, P. Fourcroy, J.-C. Davidian, F. W. Smith, and 1 metallothionein cDNAs in Vicia faba,” Plant Molecular M. J. Hawkesford, “Cloning of two contrasting high-affinity Biology, vol. 33, no. 4, pp. 583–591, 1997. sulfate transporters from tomato induced by low sulfate [39] A. Murphy, J. Zhou, P. B. Goldsbrough, and L. Taiz, “Purifi- and infection by the vascular pathogen Verticillium dahliae,” cation and immunological identification of metallothioneins Planta, vol. 218, no. 1, pp. 58–64, 2003. 1and 2from Arabidopsis thaliana,” Plant Physiology, vol. 113, [55] V. Sancenon, ´ S. Puig, H. Mira, D. J. Thiele, and L. Penar ˜ rubia, no. 4, pp. 1293–1301, 1997. “Identification of a copper transporter family in Arabidopsis [40] S. N. A. Abdullah,S.C.Cheah,and D. J. Murphy,“Isolation thaliana,” Plant Molecular Biology, vol. 51, no. 4, pp. 577–587, and characterisation of two divergent type 3 metalloth- 2003. ioneins from oil palm, Elaeis guineensis,” Plant Physiology and [56] D. Dietrich, U. Hammes, K. Thor, et al., “AtPTR1, a Biochemistry, vol. 40, no. 3, pp. 255–263, 2002. plasma membrane peptide transporter expressed during seed International Journal of Plant Genomics 13 germination and in vascular tissue of Arabidopsis,” The Plant matrix polysaccharide biosynthesis,” The Plant Journal, vol. Journal, vol. 40, no. 4, pp. 488–499, 2004. 19, no. 6, pp. 691–697, 1999. [72] A. Faik,N.J.Price,N.V.Raikhel,and K. Keegstra,“An [57] L. Maresova and H. Sychrova, “Arabidopsis thaliana CHX17 Arabidopsis gene encoding an α-xylosyltransferase involved gene complements the kha1 deletion phenotypes in Saccha- in xyloglucan biosynthesis,” Proceedings of the National romyces cerevisiae,” Yeast, vol. 23, no. 16, pp. 1167–1171, Academy of Sciences of the United States of America, vol. 99, no. 11, pp. 7797–7802, 2002. [58] M. A. Hayes, C. Davies, and I. B. Dry, “Isolation, functional [73] M. Bencur ´ ova, ´ D. Rendic, ´ G. Fabini, E.-M. Kopecky, F. characterization, and expression analysis of grapevine (Vitis Altmann, and I. B. H. Wilson, “Expression of eukaryotic vinifera L.) hexose transporters: differential roles in sink and glycosyltransferases in the yeast Pichia pastoris,” Biochimie, source tissues,” Journal of Experimental Botany, vol. 58, no. 8, vol. 85, no. 3-4, pp. 413–422, 2003. pp. 1985–1997, 2007. [74] R.-H. Fu, A.-Y. Wang, Y.-C. Wang, and H.-Y. Sung, “A cDNA [59] S. Schneider, A. Schneidereit, P. Udvardi, et al., “Arabidopsis encoding vacuolar type β-D-fructofuranosidase (Osβfruct3) Inositol Transporter2 mediates H symport of different inos- of rice and its expression in Pichia pastoris,” Biotechnology itol epimers and derivatives across the plasma membrane,” Letters, vol. 25, no. 18, pp. 1525–1530, 2003. Plant Physiology, vol. 145, no. 4, pp. 1395–1407, 2007. [75] N. Nourizad, M. Ehn, B. Gharizadeh, S. Hober, and P. Nyren, ´ [60] A. Meyer, S. Eskandari, S. Grallath, and D. Rentsch, “AtGAT1, “Methylotrophic yeast Pichia pastoris as a host for production a high affinity transporter for γ-aminobutyric acid in of ATP-diphosphohydrolase (apyrase) from potato tubers Arabidopsis thaliana,” The Journal of Biological Chemistry, vol. (Solanum tuberosum),” Protein Expression and Purification, 281, no. 11, pp. 7197–7204, 2006. vol. 27, no. 2, pp. 229–237, 2003. [61] D. Loque, ´ U. Ludewig, L. Yuan, and N. von Wiren, ´ “Tono- [76] M. M. Whittaker and J. W. Whittaker, “Characterization plast intrinsic proteins AtTIP2;1 and AtTIP2;3 facilitate NH of recombinant barley oxalate oxidase expressed by Pichia transport into the vacuole,” Plant Physiology, vol. 137, no. 2, pastoris,” Journal of Biological Inorganic Chemistry, vol. 7, no. pp. 671–680, 2005. 1-2, pp. 136–145, 2002. [62] M. Ramsperger-Gleixner, D. Geiger, R. Hedrich, and N. [77] H.-Y. Pan, M. M. Whittaker, R. Bouveret, A. Berna, F. Bernier, Sauer, “Differential expression of sucrose transporter and and J. W. Whittaker, “Characterization of wheat germin polyol transporter genes during maturation of common (oxalate oxidase) expressed by Pichia pastoris,” Biochemical plantain companion cells,” Plant Physiology, vol. 134, no. 1, and Biophysical Research Communications, vol. 356, no. 4, pp. pp. 147–160, 2004. 925–929, 2007. [63] R. Madrid, M. J. Gomez, ´ J. Ramos, and A. Rodr´ ıguez- [78] B. Kaplan, S. Tunca, and Z. Sayers, “Expression of A. thaliana Navarro, “Ectopic potassium uptake in trk1 trk2 mutants G protein alpha subunit in P. pastoris,” FEBS Journal, vol. 272, of Saccharomyces cerevisiae correlates with a highly hyper- supplement 1, pp. 1–11, 2005. polarized membrane potential,” The Journal of Biological [79] N. Lannoo, W. Vervecken, P. Proost, P. Rouge, ´ and E. J. M. Chemistry, vol. 273, no. 24, pp. 14838–14844, 1998. Van Damme, “Expression of the nucleocytoplasmic tobacco [64] J. L. Cereghino and J. M. Cregg, “Heterologous protein lectin in the yeast Pichia pastoris,” Protein Expression and expression in the methylotrophic yeast Pichia pastoris,” FEMS Purification, vol. 53, no. 2, pp. 275–282, 2007. Microbiology Reviews, vol. 24, no. 1, pp. 45–66, 2000. [80] E. Diatloff,B.G.Forde,and S. K. Roberts, “Expression and transport characterisation of the wheat low-affinity cation [65] S. Macauley-Patrick,M.L.Fazenda,B.McNeil, andL.M. transporter (LCT1) in the methylotrophic yeast Pichia pas- Harvey, “Heterologous protein production using the Pichia toris,” Biochemical and Biophysical Research Communications, pastoris expression system,” Yeast, vol. 22, no. 4, pp. 249–270, vol. 344, no. 3, pp. 807–813, 2006. [81] D. L. Andrews, B. Beames, M. D. Summers, and W. D. Park, [66] L. S. Grinna and J. F. Tschopp, “Size distribution and general “Characterization of the lipid acyl hydrolase activity of the structural features of N-linked oligosaccharides from the major potato (Solanum tuberosum) tuber protein, patatin, by methylotrophic yeast, Pichia pastoris,” Yeast,vol. 5, no.2,pp. cloning and abundant expression in a baculovirus vector,” 107–115, 1989. Biochemical Journal, vol. 252, no. 1, pp. 199–206, 1988. [67] J. M. Cregg, T. S. Vedvick, and W. C. Raschke, “Recent [82] M. Mizutani and D. Ohta, “Two isoforms of NAPDH: advances in the expression of foreign genes in Pichia pastoris,” cytochrome p450 reductase in Arabidopsis thaliana gene Nature Biotechnology, vol. 11, no. 8, pp. 905–910, 1993. structure, heterologous expression in insect cells, and differ- [68] R. K. Bretthauer and F. J. Castellino, “Glycosylation of ential regulation,” Plant Physiology, vol. 116, no. 1, pp. 357– Pichia pastoris-derived proteins,” Biotechnology and Applied 367, 1998. Biochemistry, vol. 30, no. 3, pp. 193–200, 1999. [83] H. Hayashi, L. De Bellis, A. Ciurli, M. Kondo, M. Hayashi, [69] J. A. Mertens, N. Shiraishi, and W. H. Campbell, “Recom- and M. Nishimura, “A novel Acyl-CoA oxidase that can binant expression of molybdenum reductase fragments of oxidize short-chain Acyl-CoA in plant peroxisomes,” The plant nitrate reductase at high levels in Pichia pastoris,” Plant Journal of Biological Chemistry, vol. 274, no. 18, pp. 12715– Physiology, vol. 123, no. 2, pp. 743–756, 2000. 12721, 1999. [70] W.-C. Huang, A.-Y. Wang, L.-T. Wang, and H.-Y. Sung, [84] H. Harashima, A. Shinmyo, and M. Sekine, “Phosphoryla- “Expression and characterization of sweet potato invertase in tion of threonine 161 in plant cyclin-dependent kinase A Pichia pastoris,” Journal of Agricultural and Food Chemistry, is required for cell division by activation of its associated vol. 51, no. 5, pp. 1494–1499, 2003. kinase,” The Plant Journal, vol. 52, no. 3, pp. 435–448, 2007. [71] M. E. Edwards, C. A. Dickson, S. Chengappa, C. Sidebottom, [85] M. Fukuchi-Mizutani, M. Mizutani, Y. Tanaka, T. Kusumi, M. J. Gidley, and J. S. G. Reid, “Molecular characterisation and D. Ohta, “Microsomal electron transfer in higher plants: of a membrane-bound galactosyltransferase of plant cell wall cloning and heterologous expression of NADH-cytochrome 14 International Journal of Plant Genomics b reductase from Arabidopsis,” Plant Physiology, vol. 119, no. Nutrition and Food Research, vol. 49, no. 3, pp. 228–234, 1, pp. 353–361, 1999. 2005. [86] D. Caldelari, H. Sternberg, M. Rodr´ ıguez-Concepcion, ´ W. [101] K. J. Boorer, B. G. Forde, R. A. Leigh, and A. J. Miller, “Func- Gruissem, and S. Yalovsky, “Efficient prenylation by a plant tional expression of a plant plasma membrane transporter in geranylgeranyltransferase-I requires a functional Caal box Xenopus oocytes,” FEBS Letters, vol. 302, no. 2, pp. 166–168, motif and a proximal polybasic domain,” Plant Physiology, 1992. vol. 126, no. 4, pp. 1416–1429, 2001. [102] M. Orsel, F. Chopin, O. Leleu, et al., “Characterization [87] H. Hayashi, L. De Bellis, Y. Hayashi, et al., “Molecular char- of a two-component high-affinity nitrate uptake system acterization of an Arabidopsis acyl-coenzyme a synthetase in Arabidopsis. Physiology and protein-protein interaction,” localized on glyoxysomal membranes,” Plant Physiology, vol. Plant Physiology, vol. 142, no. 3, pp. 1304–1317, 2006. 130, no. 4, pp. 2019–2026, 2002. [103] W. Liu, D. J. Fairbairn, R. J. Reid, and D. P. Schachtman, [88] B. Savidge, J. D. Weiss, Y.-H. H. Wong, et al., “Isolation and “Characterization of two HKT1 homologues from Eucalyptus characterization of homogentisate phytyltransferase genes camaldulensis that display intrinsic osmosensing capability,” from Synechocystis sp. PCC 6803 and Arabidopsis,” Plant Plant Physiology, vol. 127, no. 1, pp. 283–294, 2001. Physiology, vol. 129, no. 1, pp. 321–332, 2002. [104] P. Maser ¨ , Y. Hosoo, S. Goshima, et al., “Glycine residues [89] S. Saito, N. Hirai, C. Matsumoto, et al., “Arabidopsis in potassium channel-like selectivity filters determine potas- CYP707As encode (+)-abscisic acid 8 -hydroxylase, a key sium selectivity in four-loop-per-subunit HKT transporters enzyme in the oxidative catabolism of abscisic acid,” Plant from plants,” Proceedings of the National Academy of Sciences Physiology, vol. 134, no. 4, pp. 1439–1449, 2004. of the United States of America, vol. 99, no. 9, pp. 6428–6433, [90] S. Pagny, F. Bouissonnie, M. Sarkar, et al., “Structural 2002. requirements for Arabidopsis β1, 2-xylosyltransferase activ- [105] U. Ludewig, S. Wilken, B. Wu, et al., “Homo- and het- ity and targeting to the Golgi,” The Plant Journal, vol. 33, no. erooligomerization of ammonium transporter-1 NH uni- 1, pp. 189–203, 2003. porters,” The Journal of Biological Chemistry, vol. 278, no. 46, pp. 45603–45610, 2003. [91] N. Furman-Matarasso, E. Cohen, Q. Du, N. Chejanovsky, U. Hanania, and A. Avni, “A point mutation in the ethylene- [106] B. Neuhauser ¨ , M. Dynowski, M. Mayer, and U. Ludewig, inducing xylanase elicitor inhibits the β-1-4-endoxylanase “Regulation of NH transport by essential cross talk between activity but not the elicitation activity,” Plant Physiology, vol. AMT monomers through the carboxyl tails,” Plant Physiol- 121, no. 2, pp. 345–351, 1999. ogy, vol. 143, no. 4, pp. 1651–1659, 2007. [92] D. N. P. Doan, H. Rudi, and O.-A. Olsen, “The allosterically [107] D. Chandran, A. Reinders, and J. M. Ward, “Substrate unregulated isoform of ADP-glucose pyrophosphorylase specificity of the Arabidopsis thaliana sucrose transporter from barley endosperm is the most likely source of ADP- AtSUC2,” The Journal of Biological Chemistry, vol. 278, no. glucose incorporated into endosperm starch,” Plant Physiol- 45, pp. 44320–44325, 2003. ogy, vol. 121, no. 3, pp. 965–975, 1999. [108] A. B. Sivitz, A. Reinders, M. E. Johnson, et al., “Arabidopsis [93] F. Gaymard, M. Cerutti, C. Horeau, et al., “The bac- sucrose transporter AtSUC9. High-affinity transport activity, ulovirus/insect cell system as an alternative to Xenopus intragenic control of expression, and early flowering mutant oocytes. First characterization of the AKT1 K channel from phenotype,” Plant Physiology, vol. 143, no. 1, pp. 188–198, Arabidopsis thaliana,” The Journal of Biological Chemistry, vol. 2007. 271, no. 37, pp. 22863–22870, 1996. [109] A. Reinders, A. B. Sivitz, C. G. Starker, J. S. Gantt, and J. [94] I. Marten,F.Gaymard,G.Lemaillet,J.-B. Thibaud, H. M. Ward, “Functional analysis of LjSUT4, a vacuolar sucrose Sentenac, and R. Hedrich, “Functional expression of the transporter from Lotus japonicus,” Plant Molecular Biology, plant K channel KAT1 in insect cells,” FEBS Letters, vol. 380, vol. 68, no. 3, pp. 289–299, 2008. no. 3, pp. 229–232, 1996. [110] T. Sasaki, Y. Yamamoto, B. Ezaki, et al., “A wheat gene [95] K. Czempinski, S. Zimmermann, T. Ehrhardt, and B. Muller ¨ - encoding an aluminum-activated malate transporter,” The Rober ¨ , “New structure and function in plant K channels: Plant Journal, vol. 37, no. 5, pp. 645–653, 2004. 2+ KCO1, an outward rectifier with a steep Ca dependency,” [111] A. Ligaba, M. Katsuhara, P. R. Ryan, M. Shibasaka, and The EMBO Journal, vol. 16, no. 10, pp. 2565–2575, 1997. H. Matsumoto, “The BnALMT1 and BnALMT2 genes from [96] T. Ehrhardt, S. Zimmermann, and B. Muller ¨ -Rober ¨ , “Associ- rape encode aluminum-activated malate transporters that ation of plant K (in) channels is mediated by conserved and enhance the aluminum resistance of plant cells,” Plant does not affect subunit assembly,” FEBS Letters, vol. 409, no. Physiology, vol. 142, no. 3, pp. 1294–1303, 2006. 2, pp. 166–170, 1997. [112] Y.-S. Klepek, D. Geiger, R. Stadler, et al., “Arabidopsis [97] S. Zimmermann, I. Talke, T. Ehrhardt, G. Nast, and B. POLYOL TRANSPORTERS, a new member of the monosac- Muller ¨ -Rob ¨ er, “Characterization of SKT1, an inwardly rec- charide transporter-like superfamily, mediates H -symport tifying potassium channel from potato, by heterologous in of numerous substrates, including myo-inositol, glycerol, and insect cells,” Plant Physiology, vol. 116, no. 3, pp. 879–890, ribose,” The Plant Cell, vol. 17, no. 1, pp. 204–218, 2005. [113] M. Ramsperger-Gleixner, D. Geiger, R. Hedrich, and N. [98] D. J. Carrier, N. T. A. Bakar, R. Swarup, et al., “The binding Sauer, “Differential expression of sucrose transporter and of auxin to the Arabidopsis auxin influx transporter AUX1,” polyol transporter genes during maturation of common Plant Physiology, vol. 148, no. 1, pp. 529–535, 2008. plantain companion cells,” Plant Physiology, vol. 134, no. 1, pp. 147–160, 2004. [99] A. J. Miller and J. J. Zhou, “Xenopus oocytes as an expression system for plant transporters,” Biochimica et Biophysica Acta, [114] S. Schneider, A. Schneidereit, K. R. Konrad, et al., “Arabidop- vol. 1465, no. 1-2, pp. 343–358, 2000. sis INOSITOL TRANSPORTER4 mediates high-affinity H [100] E. Sigel, “The Xenopus oocyte: system for the study of func- symport of myoinositol across the plasma membrane,” Plant tional expression and modulation of proteins,” Molecular Physiology, vol. 141, no. 2, pp. 567–577, 2006. International Journal of Plant Genomics 15 [115] S. Schneider, A. Schneidereit, P. Udvardi, et al., “Arabidop- for N-glycosylation of the pro-region,” The Journal of Biolog- sis INOSITOL TRANSPORTER2 mediates H symport of ical Chemistry, vol. 265, no. 27, pp. 16661–16666, 1990. different inositol epimers and derivatives across the plasma [131] K. L. Korth and C. S. Levings III, “Baculovirus expression of membrane,” Plant Physiology, vol. 145, no. 4, pp. 1395–1407, the maize mitochondrial protein URF13 confers insecticidal 2007. activity in cell cultures and larvae,” Proceedings of the [116] U. Z. Hammes, E. Nielsen, L. A. Honaas, C. G. Taylor, and D. National Academy of Sciences of the United States of America, vol. 90, no. 8, pp. 3388–3392, 1993. P. Schachtman, “AtCAT6, a sink-tissue-localized transporter for essential amino acids in Arabidopsis,” The Plant Journal, [132] J. Muschietti, L. Dircks, G. Vancanneyt, and S. McCormick, “LAT52 protein is essential for tomato pollen development: vol. 48, no. 3, pp. 414–426, 2006. pollen expressing antisense LAT52 RNA hydrates and germi- [117] J. M. Colmenero-Flores, G. Mart´ ınez, G. Gamba, et al., nates abnormally and cannot achieve fertilization,” The Plant “Identification and functional characterization of cation- Journal, vol. 6, no. 3, pp. 321–328, 1994. chloride cotransporters in plants,” The Plant Journal, vol. 50, [133] H. MacDonald, J. Henderson, R. M. Napier, M. A. Venis, no. 2, pp. 278–292, 2007. C. Hawes, and C. M. Lazarus, “Authentic processing and [118] M. A. Piner ˇ os, G. M. A. Canc¸ado,L.G.Maron,S.M.Lyi, targeting of active maize auxin-binding protein in the M. Menossi, and L. V. Kochian, “Not all ALMT1-type trans- baculovirus expression system,” Plant Physiology, vol. 105, porters mediate aluminum-activated organic acid responses: no. 4, pp. 1049–1057, 1994. the case of ZmALMT1—an anion-selective transporter,” The [134] J. M. Bauly, I. M. Sealy, H. Macdonald, et al., “Overexpression Plant Journal, vol. 53, no. 2, pp. 352–367, 2008. of auxin-binding protein enhances the sensitivity of guard [119] H. Sentenac, N. Bonneaud, M. Minet, et al., “Cloning and cells to auxin,” Plant Physiology, vol. 124, no. 3, pp. 1229– expression in yeast of a plant potassium ion transport 1238, 2000. system,” Science, vol. 256, no. 5057, pp. 663–665, 1992. + [135] H. Y. Meller Harel, V. Fontaine, H. Chen, I. M. Jones, and P. [120] I. Dreyer, S. Antunes, T. Hoshi, et al., “Plant K channel α- A. Millner, “Display of a maize cDNA library on baculovirus subunits assemble indiscriminately,” Biophysical Journal, vol. infected insect cells,” BMC Biotechnology, vol. 8, pp. 64–69, 72, no. 5, pp. 2143–2150, 1997. [121] P. H. Buschmann, R. Vaidyanathan, W. Gassmann, and J. + [136] M. Mizutani, D. Ohta, and R. Sato, “Isolation of a cDNA I. Schroeder, “Enhancement of Na uptake currents, time- and a genomic clone encoding cinnamate 4-hydroxylase + + dependent inward-rectifying K channel currents, and K + from Arabidopsis and its expression manner in planta,” Plant channel transcripts by K starvation in wheat root cells,” Physiology, vol. 113, no. 3, pp. 755–763, 1997. Plant Physiology, vol. 122, no. 4, pp. 1387–1397, 2000. [137] J.-P. Bouly, B. Giovani, A. Djamei, et al., “Novel ATP- [122] E. Formentin, S. Varotto, A. Costa, et al., “DKT1, a novel K binding and autophosphorylation activity associated with channel from carrot, forms functional heteromeric channels Arabidopsis and human cryptochrome-1,” European Journal with KDC1,” FEBS Letters, vol. 573, no. 1–3, pp. 61–67, 2004. of Biochemistry, vol. 270, no. 14, pp. 2921–2928, 2003. [123] I. Fuchs, S. Stolzle, ¨ N. Ivashikina, and R. Hedrich, “Rice K [138] H.-Y. Cho, T.-S. Tseng, E. Kaiserli, S. Sullivan, J. M. Christie, uptake channel OsAKT1 is sensitive to salt stress,” Planta, vol. and W. R. Briggs, “Physiological roles of the light, oxygen, 221, no. 2, pp. 212–221, 2005. or voltage domains of phototropin 1 and phototropin 2 in [124] V. Van Wilder, U. Miecielica, H. Degand, R. Derua, E. Arabidopsis,” Plant Physiology, vol. 143, no. 1, pp. 517–529, Waelkens, and F. Chaumont, “Maize plasma membrane aquaporins belonging to the PIP1 and PIP2 subgroups are in [139] A. Nagai, K. Suzuki, E. Ward, et al., “Overexpression of plant vivo phosphorylated,” Plant and Cell Physiology, vol. 49, no. histidinol dehydrogenase using a baculovirus expression 9, pp. 1364–1377, 2008. vector system,” Archives of Biochemistry and Biophysics, vol. [125] D. C. Bassham and N. V. Raikhel, “Plant cells are not just 295, no. 2, pp. 235–239, 1992. green yeast,” Plant Physiology, vol. 122, no. 4, pp. 999–1001, [140] A. Stapleton, J. K. Beetham, F. Pinot, et al., “Cloning and expression of soluble epoxide hydrolase from potato,” The [126] X. Dong, B. Tang, J. Li, Q. Xu, S. Fang, and Z. Hua, Plant Journal, vol. 6, no. 2, pp. 251–258, 1994. “Expression and purification of intact and functional soy- [141] S. Tada, M. Hatano, Y. Nakayama, et al., “Insect cell expres- bean (Glycine max) seed ferritin complex in Escherichia coli,” sion of recombinant imidazoleglycerolphosphate dehy- Journal of Microbiology and Biotechnology,vol. 18, no.2,pp. dratase of Arabidopsis and wheat and inhibition by triazole 299–307, 2008. herbicides,” Plant Physiology, vol. 109, no. 1, pp. 153–159, [127] J. Lin, R. Fido, P. Shewry, D. B. Archer, and M. J. C. Alcocer, “The expression and processing of two recombinant 2S [142] M. Schledz, S. Al-Babili, J. von Lintig, et al., “Phytoene albumins from soybean (Glycine max) in the yeast Pichia synthase from Narcissus pseudonarcissus: functional expres- pastoris,” Biochimica et Biophysica Acta, vol. 1698, no. 2, pp. sion, galactolipid requirement, topological distribution in 203–212, 2004. chromoplasts and induction during flowering,” The Plant [128] M. M. Bustos, V. A. Luckow, L. R. Griffing,M.D.Summers, Journal, vol. 10, no. 5, pp. 781–792, 1996. and T. C. Hall, “Expression, glycosylation and secretion of [143] S. Al-Babili, J. von Lintig, H. Haubruck, and P. Beyer, “A phaseolin in a baculovirus system,” Plant Molecular Biology, novel, soluble form of phytoene desaturase from Narcis- vol. 10, no. 6, pp. 475–488, 1988. sus pseudonarcissus chromoplasts is Hsp70-complexed and [129] R. Kunze and P. Starlinger, “The putative transposase of competent for flavinylation, membrane association and transposable element Ac from Zea mays L. interacts with enzymatic activation,” The Plant Journal,vol. 9, no.5,pp. subterminal sequences of Ac,” The EMBO Journal, vol. 8, no. 601–612, 1996. 11, pp. 3177–3185, 1989. [144] S. M. Allina, A. Pri-Hadash, D. A. Theilmann, B. E. Ellis, [130] T. Vernet, D. C. Tessier, C. Richardson, et al., “Secretion of and C. J. Douglas, “4-coumarate:coenzyme a ligase in hybrid functional papain precursor from insect cells. Requirement poplar. Properties of native enzymes, cDNA cloning, and 16 International Journal of Plant Genomics analysis of recombinant enzymes,” Plant Physiology, vol. 116, parasite Cuscuta reflexa,” Planta, vol. 213, no. 4, pp. 550–555, no. 2, pp. 743–754, 1998. 2001. [145] R. Pratelli, B. Lacombe, L. Torregrosa, et al., “A grapevine [159] S. Sakr, G. Alves, R. Morillon, et al., “Plasma membrane gene encoding a guard cell K channel displays developmen- aquaporins are involved in winter embolism recovery in tal regulation in the grapevine berry,” Plant Physiology, vol. walnut tree,” Plant Physiology, vol. 133, no. 2, pp. 630–641, 128, no. 2, pp. 564–577, 2002. 2003. [146] K. Philippar, K. Buc ¨ hsenschutz, ¨ M. Abshagen, et al., “The K [160] L.-H. Liu, U. Ludewig, B. Gassert, W. B. Frommer, and N. channel KZM1 mediates potassium uptake into the phloem von Wiren, ´ “Urea transport by nitrogen-regulated tonoplast and guard cells of the C grass Zea mays,” The Journal of intrinsic proteins in Arabidopsis,” Plant Physiology, vol. 133, Biological Chemistry, vol. 278, no. 19, pp. 16973–16981, 2003. no. 3, pp. 1220–1228, 2003. [147] K. Philippar, I. Fuchs, H. Luthen, ¨ et al., “Auxin-induced K [161] D. Loque, ´ U. Ludewig, L. Yuan, and N. von Wiren, ´ “Tono- channel expression represents an essential step in coleop- plast intrinsic proteins AtTIP2;1 and AtTIP2;3 facilitate NH tile growth and gravitropism,” Proceedings of the National transport into the vacuole,” Plant Physiology, vol. 137, no. 2, Academy of Sciences of the United States of America, vol. 96, pp. 671–680, 2005. no. 21, pp. 12186–12191, 1999. [162] R. Vera-Estrella, B. J. Barkla, H. J. Bohnert, and O. Pantoja, [148] S. Hartje, S. Zimmermann, D. Klonus, and B. Mueller- “Novel regulation of aquaporins during osmotic stress,” Plant Roeber, “Functional characterisation of LKT1, a K uptake Physiology, vol. 135, no. 4, pp. 2318–2329, 2004. channel from tomato root hairs, and comparison with the [163] W. Wei, E. Alexandersson, D. Golldack, A. J. Miller, P. O. closely related potato inwardly rectifying K channel SKT1 Kjellbom, and W. Fricke, “HvPIP1;6, a barley (Hordeum after expression in Xenopus oocytes,” Planta, vol. 210, no. 5, vulgare L.) plasma membrane water channel particularly pp. 723–731, 2000. expressed in growing compared with non-growing leaf [149] J. Xicluna, B. Lacombe, I. Dreyer, et al., “Increased functional tissues,” Plant and Cell Physiology, vol. 48, no. 8, pp. 1132– diversity of plant K channels by preferential heteromer- 1147, 2007. ization of the Shaker-like subunits AKT2 and KAT2,” The [164] W. Lin, Y. Peng, G. Li, et al., “Isolation and functional char- Journal of Biological Chemistry, vol. 282, no. 1, pp. 486–494, acterization of PgTIP1, a hormone-autotrophic cells-specific tonoplast aquaporin in ginseng,” Journal of Experimental [150] B. Sottocornola, S. Visconti, S. Orsi, et al., “The potassium Botany, vol. 58, no. 5, pp. 947–956, 2007. channel KAT1 is activated by plant and animal 14-3-3 [165] W.-G. Choi and D. M. Roberts, “Arabidopsis NIP2;1, a major proteins,” The Journal of Biological Chemistry, vol. 281, no. intrinsic protein transporter of lactic acid induced by anoxic 47, pp. 35735–35741, 2006. stress,” The Journal of Biological Chemistry, vol. 282, no. 33, [151] Q. Leng, R. W. Mercier, B.-G. Hua, H. Fromm, and G. pp. 24209–24218, 2007. A. Berkowitz, “Electrophysiological analysis of cloned cyclic [166] M. Mahdieh, A. Mostajeran, T. Horie, and M. Katsuhara, nucleotide-gated ion channels,” Plant Physiology, vol. 128, “Drought stress alters water relations and expression of PIP- no. 2, pp. 400–410, 2002. type aquaporin genes in Nicotiana tabacum plants,” Plant and [152] B.-G. Hua, R. W. Mercier, Q. Leng, and G. A. Berkowitz, Cell Physiology, vol. 49, no. 5, pp. 801–813, 2008. “Plants do it differently. A new basis for potassium/sodium [167] N. Shitan, I. Bazin, K. Dan, et al., “Involvement of CjMDR1, selectivity in the pore of an ion channel,” Plant Physiology, a plant multidrugresistance-type ATP-binding cassette pro- vol. 132, no. 3, pp. 1353–1361, 2003. tein, in alkaloid transport in Coptis japonica,” Proceedings [153] E. D. Vincill, K. Szczyglowski, and D. M. Roberts, “GmN70 of the National Academy of Sciences of the United States of and LjN70. Anion transporters of the symbiosome mem- America, vol. 100, no. 2, pp. 751–756, 2003. brane of nodules with a transport preference for nitrate,” [168] S. Gustavsson, A.-S. Lebrun, K. Norden, ´ F. Chaumont, Plant Physiology, vol. 137, no. 4, pp. 1435–1444, 2005. and U. Johanson, “A novel plant major intrinsic protein [154] M. A. Piner ˜ os, G. M. A. Canc ¸ ado, and L. V. Kochian, in Physcomitrella patens most similar to bacterial glycerol “Novel properties of the wheat aluminum tolerance organic channels,” Plant Physiology, vol. 139, no. 1, pp. 287–295, acid transporter (TaALMT1) revealed by electrophysiological 2005. characterization in Xenopus oocytes: functional and struc- [169] A.-G. Desbrosses-Fonrouge, K. Voigt, A. Schroder ¨ , S. tural implications,” Plant Physiology, vol. 147, no. 4, pp. Arrivault, S. Thomine, and U. Kramer ¨ , “Arabidopsis thaliana 2131–2146, 2008. MTP1 is a Zn transporter in the vacuolar membrane which [155] A. Meyer, S. Eskandari, S. Grallath, and D. Rentsch, “AtGAT1, mediates Zn detoxification and drives leaf Zn accumulation,” a high affinity transporter for γ-aminobutyric acid in FEBS Letters, vol. 579, no. 19, pp. 4165–4174, 2005. Arabidopsis thaliana,” The Journal of Biological Chemistry, vol. [170] D. Becker, D. Geiger, M. Dunkel, et al., “AtTPK4, an 281, no. 11, pp. 7197–7204, 2006. Arabidopsis tandem-pore K channel, poised to control the 2+ [156] F. Chaumont, F. Barrieu, R. Jung, and M. J. Chrispeels, pollen membrane voltage in a pH- and Ca -dependent “Plasma membrane intrinsic proteins from maize cluster in manner,” Proceedings of the National Academy of Sciences of two sequence subgroups with differential aquaporin activity,” the United States of America, vol. 101, no. 44, pp. 15621– Plant Physiology, vol. 122, no. 4, pp. 1025–1034, 2000. 15626, 2004. [157] C. Dordas, M. J. Chrispeels, and P. H. Brown, “Permeability [171] T. P. Durrett, W. Gassmann, and E. E. Rogers, “The FRD3- and channel-mediated transport of boric acid across mem- mediated efflux of citrate into the root vasculature is brane vesicles isolated from squash roots,” Plant Physiology, necessary for efficient iron translocation,” Plant Physiology, vol. 124, no. 3, pp. 1349–1361, 2000. vol. 144, no. 1, pp. 197–205, 2007. [158] M. Werner, N. Uehlein, P. Proksch, and R. Kaldenhoff, “Characterization of two tomato aquaporins and expression during the incompatible interaction of tomato with the plant International Journal of Peptides Advances in International Journal of BioMed Stem Cells Virolog y Research International International Genomics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Nucleic Acids International Journal of Zoology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com The Scientific Journal of Signal Transduction World Journal Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Advances in Genetics Anatomy Biochemistry Research International Research International Microbiology Research International Bioinformatics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Enzyme Journal of International Journal of Molecular Biology Archaea Research Evolutionary Biology International Marine Biology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Journal

International Journal of Plant GenomicsHindawi Publishing Corporation

Published: Aug 6, 2009

References