SECRETION AND INVIVO FOLDING OF THE FAB FRAGMENT OF THE ANTIBODY MCPC603 IN ESCHERICHIA-COLI - INFLUENCE OF DISULFIDES AND CIS-PROLINES

被引:86
作者
SKERRA, A [1 ]
PLUCKTHUN, A [1 ]
机构
[1] UNIV MUNICH, MAX PLANCK INST BIOCHEM, GENZENTRUM, AM KLOPFERSPITZ, W-8033 MARTINSRIED, GERMANY
来源
PROTEIN ENGINEERING | 1991年 / 4卷 / 08期
关键词
ANTIBODY; EXPRESSION IN ESCHERICHIA-COLI; PROTEIN FOLDING; PROTEIN SECRETION;
D O I
10.1093/protein/4.8.971
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Using the well-characterized antibody McPC603 as a model, we had found that the F(v) fragment can be isolated from Escherichia coli as a functional protein in good yields, whereas the amount of the correctly folded F(ab) fragment of the same antibody produced under identical conditions is significantly lower. In this paper, we analyse the reasons for this difference. We found that a variety of signal sequences function in the secretion of the isolated chains of the F(ab) fragment or in the co-secretion of both chains in E. coli. The low yield of functional F(ab) fragment is not caused by inefficient expression or secretion in E. coli, but by inefficient folding and/or assembly in the periplasm. We compared the folding yields for the F(v) and the F(ab) fragment in the periplasm under various conditions. Several diagnostic framework variants were constructed and their folding yields measured. The results show that substitutions affecting cis-proline residues and those affecting various disulphide bonds in the protein are by themselves insufficient to dramatically change the partitioning of the folding pathway to the native structure, and the cause must lie in a facile aggregation of folding intermediates common to all structural variants. However, all structural variants could be obtained in native form, demonstrating the general utility of the secretory expression strategy.
引用
收藏
页码:971 / 979
页数:9
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