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Increasing the soluble expression and crystallization of the Escherichia coli quorum‐sensing protein LsrK

Increasing the soluble expression and crystallization of the Escherichia coli quorum‐sensing... LsrK is one of the key components of the luxS‐regulated (lsr) operon in Escherichia coli and plays an important role during the quorum‐sensing (QS) process mediated by autoinducer‐2 (AI‐2). The AI‐2 molecule is imported into the cell by the LsrACB transporter and is subsequently phosphorylated (to AI‐2‐P) by LsrK. AI‐2‐P binds to the repressor protein of the lsr operon (LsrR) and triggers various cellular responses related to QS by dissociating LsrR from the DNA. Although a large amount of purified LsrK is required for structural studies, recombinant GST‐LsrK was mostly expressed in an insoluble form. To enhance the soluble expression of LsrK, an attempt was made to increase the expression of the cellular chaperone proteins that are well known to support proper protein folding. Transformed E. coli was cultured in high‐salt LB medium and heat shock was applied prior to subsequent IPTG induction at 20°C. These procedures increased the yield of purified LsrK by about tenfold compared with standard IPTG induction at 20°C. The expressed LsrK was readily purified by GST‐affinity chromatography. Crystals of LsrK were grown by the hanging‐drop vapour‐diffusion method. The X‐ray diffraction data of the crystal were processed in a primitive hexagonal space group to 2.9 Å resolution. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Crystallographica Section F Wiley

Increasing the soluble expression and crystallization of the Escherichia coli quorum‐sensing protein LsrK

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References (15)

Publisher
Wiley
Copyright
Copyright © 2017 Wiley Subscription Services
ISSN
2053-230X
eISSN
2053-230X
DOI
10.1107/S2053230X1700468X
pmid
28471356
Publisher site
See Article on Publisher Site

Abstract

LsrK is one of the key components of the luxS‐regulated (lsr) operon in Escherichia coli and plays an important role during the quorum‐sensing (QS) process mediated by autoinducer‐2 (AI‐2). The AI‐2 molecule is imported into the cell by the LsrACB transporter and is subsequently phosphorylated (to AI‐2‐P) by LsrK. AI‐2‐P binds to the repressor protein of the lsr operon (LsrR) and triggers various cellular responses related to QS by dissociating LsrR from the DNA. Although a large amount of purified LsrK is required for structural studies, recombinant GST‐LsrK was mostly expressed in an insoluble form. To enhance the soluble expression of LsrK, an attempt was made to increase the expression of the cellular chaperone proteins that are well known to support proper protein folding. Transformed E. coli was cultured in high‐salt LB medium and heat shock was applied prior to subsequent IPTG induction at 20°C. These procedures increased the yield of purified LsrK by about tenfold compared with standard IPTG induction at 20°C. The expressed LsrK was readily purified by GST‐affinity chromatography. Crystals of LsrK were grown by the hanging‐drop vapour‐diffusion method. The X‐ray diffraction data of the crystal were processed in a primitive hexagonal space group to 2.9 Å resolution.

Journal

Acta Crystallographica Section FWiley

Published: Jan 1, 2017

Keywords: ; ; ; ; ; ;

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