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The Functional Expression of Antibody Fv Fragments in Ischhuchia coli: Improved Vectors and a Generally Applicable Purification Technique

Bio/Technology volume 9, pages 273–278 (1991)Cite this article

Abstract

We have previously demonstrated that the expression of fully functional Fv and Fab fragments in E. coli is possible by the simultaneous secretion of both chains to the periplasm. To increase production levels and facilitate engineering and random mutagenesis, we improved our previous vectors by introducing a resident represser gene and a filamentous phage origin. We also developed a new purification strategy based on immobilized metal ion chromatography, with which a single-chain Fv fragment can be purified to homogeneity in a single step. We investigated the most efficient tail constructions and found that only a minimal structural change of three additional C-terminal amino acids is necessary. This modification has no deleterious effect on in vivo transport and folding or antigen affinity.

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Author notes

  1. Arne Skerra

    Present address: Abt. Molekulare Membranbiologie, Max-Planck-Institut für Biophysik, D-6000, Frankfurt, 70, FRG

  2. Andreas Plückthun: Corresponding author.

Authors and Affiliations

  1. Genzentrum der Universität Munchen, Max-Planck-Institut für Biochemie, D-8033, Martinsried, FRG

    Ilse Pfitzinger & Andreas Plückthun

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  1. Arne Skerra
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  2. Ilse Pfitzinger
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  3. Andreas Plückthun
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Skerra, A., Pfitzinger, I. & Plückthun, A. The Functional Expression of Antibody Fv Fragments in Ischhuchia coli: Improved Vectors and a Generally Applicable Purification Technique. Nat Biotechnol 9, 273–278 (1991). https://doi.org/10.1038/nbt0391-273

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