Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv

Appl Microbiol Biotechnol. 2015 Mar;99(6):2693-703. doi: 10.1007/s00253-014-6268-4. Epub 2014 Dec 24.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cloning, Molecular
  • Enzyme-Linked Immunosorbent Assay
  • Freund's Adjuvant
  • Gene Expression Regulation
  • Gene Rearrangement*
  • Immunization
  • Immunoglobulin G / blood
  • Lipids
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Rabbits
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Serum Albumin
  • Single-Chain Antibodies / genetics*
  • Single-Chain Antibodies / metabolism
  • Troponin I / immunology


  • Immunoglobulin G
  • Lipids
  • Recombinant Proteins
  • Serum Albumin
  • Single-Chain Antibodies
  • Troponin I
  • incomplete Freund's adjuvant
  • Freund's Adjuvant