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

Nature Biotechnology volume 15, pages 79–84 (1997)Cite this article

Abstract

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 μg/ml in only 15 min) could be recovered from immobilized antigen.

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Author notes

  1. Lyubov A. Ryabova

    Present address: Friedrich Miescher-Institut, PO Box 2543, CH-4002, Basel, Switzerland

Authors and Affiliations

  1. Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057, Zürich, Switzerland

    Lyubov A. Ryabova, Dominique Desplancq & Andreas Plückthun

  2. Institute of Protein Research, Russian Academy of Sciences, 142292, Pushchino, Moscow region, Russia

    Lyubov A. Ryabova & Alexander S. Spirin

  3. Département des Récepteurs et Protéines Membranaires, UPR 9050 du CNRS, ESBS, Pôle Universitaire d'Illkirch, Boulevard Sébastien Brandt, F-67400, Illkirch, France

    Dominique Desplancq

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  4. Andreas Plückthun
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Correspondence to Andreas Plückthun.

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Ryabova, L., Desplancq, D., Spirin, A. et al. Functional antibody production using cell-free translation: Effects of protein disulfide isomerase and chaperones. Nat Biotechnol 15, 79–84 (1997). https://doi.org/10.1038/nbt0197-79

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