Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia coli and its "oxidizing" mutant.
OBJECTIVE
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 ED(50) of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 microg/L and 2.2 microg/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 x 10(7) 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 x 10(7) mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains.


CONCLUSIONS
Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.
Xiong
Sheng
S
Biomedical Research and Development Center, Jinan University, Guangzhou 510630, Guangdong Province, China. xiongsheng99@263.net
Wang
Yi-Fei
YF
Ren
Xiang-Rong
XR
Li
Bing
B
Zhang
Mei-Ying
MY
Luo
Yong
Y
Zhang
Ling
L
Xie
Qiu-Ling
QL
Su
Kuan-Yuan
KY
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D012995:Solubility
D000906:Antibodies; D004220:Disulfides; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2
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