Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones

被引:160
作者
Levy, R
Weiss, R
Chen, G
Iverson, BL
Georgiou, G [1 ]
机构
[1] Univ Texas, Inst Cell & Mol Biol, Austin, TX 78712 USA
[2] Univ Texas, Dept Chem Engn, Austin, TX 78712 USA
关键词
D O I
10.1006/prep.2001.1520
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Disulfide bonds are normally formed after a polypeptide has been exported from the reducing environment of the cytoplasm into a more oxidizing compartment, such as the bacterial periplasm. Recently, we showed that in Escherichia coli trxB gor mutants, in which the reduction of thioredoxin and glutathione is impaired, the redox potential of the cytoplasm becomes comparable to that of the mammalian endoplasmic reticulum, thus allowing the formation of disulfide bonds in certain complex proteins (P. H. Bessette et al., 1999, Proc. Natl. Acad. Sci. USA 96,13703-13708]. Here, we investigate the expression of a Fab antibody fragment in the bacterial cytoplasm. The effect of coexpressing cytoplasmic chaperones (GroEL/ES, trigger factor, DnaK/J), as well as signal sequenceless versions of periplasmic chaperones (DsbC and Skp), was examined. Skp coexpression was shown to have the most significant effect (five- to sixfold increase) on the yield of correctly folded Fab. A maximum yield of 0.8 mg Fab/L/OD600 Fab was obtained, indicating that cytoplasmic expression may be a viable alternative for the preparative production of antibody fragments. (C) 2001 Academic Press.
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页码:338 / 347
页数:10
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