caa a22 4500 605499292 CHVBK 20210128100550.0 cr unu---uuuuu 210128e20150301xx s 000 0 eng 10.1007/s00253-014-6268-4 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6268-4 Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv [Elektronische Daten] [B. Ayyar, Stephen Hearty, Richard O'Kennedy] 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. Springer-Verlag Berlin Heidelberg, 2014 Rabbit antibodies nationallicence scFv nationallicence scAb nationallicence Aberrant cysteine nationallicence Phage display nationallicence Ayyar B. Biomedical Diagnostics Institute, National Centre for Sensor Research, Glasnevin, Dublin City University, Dublin 9, Ireland aut Hearty Stephen Biomedical Diagnostics Institute, National Centre for Sensor Research, Glasnevin, Dublin City University, Dublin 9, Ireland aut O'Kennedy Richard Biomedical Diagnostics Institute, National Centre for Sensor Research, Glasnevin, Dublin City University, Dublin 9, Ireland aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/6(2015-03-01), 2693-2703 0175-7598 99:6<2693 2015 99 253 https://doi.org/10.1007/s00253-014-6268-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6268-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ayyar B. Biomedical Diagnostics Institute, National Centre for Sensor Research, Glasnevin, Dublin City University, Dublin 9, Ireland aut NATIONALLICENCE 700 1- Hearty Stephen Biomedical Diagnostics Institute, National Centre for Sensor Research, Glasnevin, Dublin City University, Dublin 9, Ireland aut NATIONALLICENCE 700 1- O'Kennedy Richard Biomedical Diagnostics Institute, National Centre for Sensor Research, Glasnevin, Dublin City University, Dublin 9, Ireland aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/6(2015-03-01), 2693-2703 0175-7598 99:6<2693 2015 99 253