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Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv

  • Applied genetics and molecular biotechnology
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Abstract

Rabbit-derived recombinant antibodies have traditionally been viewed as intractable molecules due to the presence of a cysteine in position 80 of the VL domain that becomes rendered ‘aberrant’ when present in the ‘unpaired’ context of a single chain Fv (scFv) and chimeric Fab formats. This aberrant Cys80 can severely impinge on the achievable expression levels when rabbit recombinant antibodies are produced in prokaryote systems. The unpaired Cys residue also renders purification problematic. Consequently, researchers often disregard rabbit antibody libraries due to perceived limitations in accessible repertoire diversity. We have shown that by switching the orientation of the VH and VL domains in an aberrant-Cys-containing rabbit scFv isolated in a bona fide screening campaign, it was possible to substantially increase the expression and purification yields of this clone. Furthermore, by incorporating a novel rabbit C-kappa constant fusion domain, we were able to potentiate a further increase in expression level and purify this antibody to a high degree of homogeneity, hitherto impossible to achieve using the aberrant-Cys-containing wild-type scFv. Cumulatively, these findings demonstrate that facile re-formatting can help make the rabbit antibody repertoire, a very valuable resource, more accessible to researchers in the field.

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Acknowledgments

The authors thank Prof. Carlos F. Barbas III (Scripps Research Institute) for kindly providing the pComb3 vector series used in this study. The authors would also like to thank Dr. Andrew Hayhurst, Department of Virology and Immunology, Texas, USA, for providing the scAb expression vector. All authors were supported by the Science Foundation Ireland under a Centres for Science Engineering and Technology (CSET) grant (05/CE3/B754).

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Author notes

  1. B. Vijayalakshmi Ayyar

    Present address: Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA

Authors and Affiliations

  1. Biomedical Diagnostics Institute, National Centre for Sensor Research, Glasnevin, Dublin City University, Dublin 9, Ireland

    B. Vijayalakshmi Ayyar, Stephen Hearty & Richard O’Kennedy

  2. School of Biotechnology, Glasnevin, Dublin City University, Dublin 9, Ireland

    B. Vijayalakshmi Ayyar, Stephen Hearty & Richard O’Kennedy

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  1. B. Vijayalakshmi Ayyar
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  2. Stephen Hearty
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  3. Richard O’Kennedy
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Corresponding author

Correspondence to Richard O’Kennedy.

Additional information

B. Vijayalakshmi Ayyar and Stephen Hearty contributed equally to this work.

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Ayyar, B.V., Hearty, S. & O’Kennedy, R. Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv. Appl Microbiol Biotechnol 99, 2693–2703 (2015). https://doi.org/10.1007/s00253-014-6268-4

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  • DOI: https://doi.org/10.1007/s00253-014-6268-4

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