Functional antibody production using cell-free translation: Effects of protein disulfide isomerase and chaperones

被引:133
作者
Ryabova, LA
Desplancq, D
Spirin, AS
Pluckthun, A
机构
[1] UNIV ZURICH,INST BIOCHEM,CH-8057 ZURICH,SWITZERLAND
[2] RUSSIAN ACAD SCI,INST PROT RES,PUSHCHINO 142292,MOSCOW REG,RUSSIA
关键词
cell-free translation system; single-chain antibody; disulfide isomerase; molecular chaperones; protein folding;
D O I
10.1038/nbt0197-79
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
To create a rapid system to test the effect of sequence changes on recombinant antibody binding, we have developed a procedure for producing functional scFv fragments in an Escherichia coli cell-free translation system. Functional antibodies with antigen-binding activity are obtained only if disulfide formation and rearrangement is allowed to take place during the translation reaction. The inclusion of protein disulfide isomerase (PDI) leads to a threefold increase in yield over that obtained in the presence of glutathione redox systems. DsbA had no such effect, indicating that disulfide shuffling, and not net formation, is the crucial yield-limiting step. The addition of the molecular chaperones DnaK and DnaJ increased the amount of soluble protein but not the amount of functional scFv, which appears to be limited entirely by correct disulfide formation. None of these factors significantly influenced total protein synthesis. In the presence of PDI, chaperones, reduced glutathione and oxidized glutathione, 50% of the scFv produced (about 8 mu g/ml in only 15 min) could be recovered from immobilized antigen.
引用
收藏
页码:79 / 84
页数:6
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