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Recombinant human J-chain: Fix the protein aggregations and yield maximize

Recombinant human J-chain: Fix the protein aggregations and yield maximize Polymeric immunoglobulin (dimeric IgA and pentameric IgM) molecules can assembly by using the immunoglobulin J (joining) chain and across the epithelial cell layers. Based on its amino acid and gene sequences data, disulfide bond (2 bonds) assignment secondary structure predictions, and chemical properties, a model for J-chain folding has been proposed. However, the crystal structure of the J-chain protein is still far from obtained, because the J-chain expression and its protein downstream has a permanent aggregation problems, due to its two free thiol groups. Our work focused on the chemical blocking of free cysteines-SH or to mutate these cysteines into serine residues. The chemical blocking yielded partially soluble proteins with new structures (carboxyamidomethyl cysteine and carboxyamidomethyl methionine) at cysteine and methionine residues. While mutate the cysteines ^{15,69} into serine has been yielded a complete soluble (11.5 mg/l) J-chain protein which migrate (SDS-PAGE) at 27 KDa. We were used pET22b expression vector and E. coli BL21 (DE3) to produce the J-chain protein. Fed-batch strategy was employed to maximize the protein concentration by using the manually predetermined exponential feeding rate. The process was automated through a computer aided data bioprocessing system AFS-BioCommand multi-process management program to regulate the cell growth rate by controlling interactively both the nutrient feed rate, temperature, pH and agitation speed based on dissolved oxygen. Maximum dry cell weight concentration in the bioreactor was 78 g/l after induction. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Antibodies IOS Press

Recombinant human J-chain: Fix the protein aggregations and yield maximize

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Publisher
IOS Press
Copyright
Copyright © 2006 by IOS Press, Inc
ISSN
1093-2607
eISSN
1875-869X
Publisher site
See Article on Publisher Site

Abstract

Polymeric immunoglobulin (dimeric IgA and pentameric IgM) molecules can assembly by using the immunoglobulin J (joining) chain and across the epithelial cell layers. Based on its amino acid and gene sequences data, disulfide bond (2 bonds) assignment secondary structure predictions, and chemical properties, a model for J-chain folding has been proposed. However, the crystal structure of the J-chain protein is still far from obtained, because the J-chain expression and its protein downstream has a permanent aggregation problems, due to its two free thiol groups. Our work focused on the chemical blocking of free cysteines-SH or to mutate these cysteines into serine residues. The chemical blocking yielded partially soluble proteins with new structures (carboxyamidomethyl cysteine and carboxyamidomethyl methionine) at cysteine and methionine residues. While mutate the cysteines ^{15,69} into serine has been yielded a complete soluble (11.5 mg/l) J-chain protein which migrate (SDS-PAGE) at 27 KDa. We were used pET22b expression vector and E. coli BL21 (DE3) to produce the J-chain protein. Fed-batch strategy was employed to maximize the protein concentration by using the manually predetermined exponential feeding rate. The process was automated through a computer aided data bioprocessing system AFS-BioCommand multi-process management program to regulate the cell growth rate by controlling interactively both the nutrient feed rate, temperature, pH and agitation speed based on dissolved oxygen. Maximum dry cell weight concentration in the bioreactor was 78 g/l after induction.

Journal

Human AntibodiesIOS Press

Published: Jan 1, 2006

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