Brief Reports
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 21, 2005; 11(7): 1077-1082
Published online Feb 21, 2005. doi: 10.3748/wjg.v11.i7.1077
Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia coli and its “oxidizing” mutant
Sheng Xiong, Yi-Fei Wang, Xiang-Rong Ren, Bing Li, Mei-Ying Zhang, Yong Luo, Ling Zhang, Qiu-Ling Xie, Kuan-Yuan Su
Sheng Xiong, Yi-Fei Wang, Bing Li, Mei-Ying Zhang, Yong Luo, Biomedical Research & Development Center, Jinan University, Guangzhou 510630, Guangdong Province, China
Xiang-Rong Ren, Kuan-Yuan Su, Center of Infectious Diseases, 458 Hospital of PLA, Guangzhou 510602, Guangdong Province, China
Ling Zhang, Qiu-Ling Xie, Institute of Bioengineering, Jinan University, Guangzhou 510630, Guangdong Province, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 30371661 and No. 30400071; and the Natural Science Foundation for Research Team of Guangdong Province, China, No. 2004E039213
Correspondence to: Dr. Sheng Xiong, Biomedical Research & Development Center, Jinan University, Guangzhou 510630, Guangdong Province, China. xiongsheng99@263.net
Telephone: +86-20-85222706 Fax: +86-20-85220504-309
Received: April 28, 2004
Revised: May 2, 2004
Accepted: June 29, 2004
Published online: February 21, 2005
Abstract

AIM: To study the influence of redox environment of Escherichia coli (E. coli) cytoplasm on disulfide bond formation of recombinant proteins.

METHODS: Bovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF, and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3) and M15[pREP4] respectively. At the same time, both plasmids were transformed into a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively.

RESULTS: All recombinant E. coli strains could efficiently produce target proteins. The level of BbFGF in BL21(DE3) was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm, and all the target proteins became soluble in Origami(DE3). The bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 μg/L and 2.2 μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami[pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv] was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from inclusion body in M15[pQE-HBscFv] was 30-35 mg/L and the affinity constant was 1.98×107 mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains.

CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.

Keywords: Escherichia coli, Recombinant protein expression, Disulfide bond, Solubility, bFGF, HBsAg, Single-chain Fv, Reductase deficient, Protein folding, Affinity constant