Ryabova L A, Desplancq D, Spirin A S, Plückthun A
Biochemisches Institut, Universität Zürich, Switzerland.
Nat Biotechnol. 1997 Jan;15(1):79-84. doi: 10.1038/nbt0197-79.
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 shuffing, 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 micrograms/ml in only 15 min) could be recovered from immobilized antigen.
为创建一个快速系统来测试序列变化对重组抗体结合的影响，我们开发了一种在大肠杆菌无细胞翻译系统中生产功能性单链抗体片段（scFv）的方法。只有当翻译反应过程中允许二硫键形成和重排时，才能获得具有抗原结合活性的功能性抗体。加入蛋白质二硫键异构酶（PDI）后，产量比在谷胱甘肽氧化还原系统存在时提高了两倍。二硫键形成蛋白A（DsbA）没有这种效果，这表明二硫键重排而非净形成是关键的产量限制步骤。添加分子伴侣DnaK和DnaJ增加了可溶性蛋白的量，但没有增加功能性scFv的量，功能性scFv的量似乎完全受正确二硫键形成的限制。这些因素均未显著影响总蛋白合成。在存在PDI、分子伴侣、还原型谷胱甘肽和氧化型谷胱甘肽的情况下，所产生的scFv中有50%（仅15分钟内约为8微克/毫升）可从固定化抗原中回收。
