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The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag)

Authors :
Ario de Marco
Aurelie Schneider
Anne Beugnet
Selma Djender
Source :
Antibodies, Vol 3, Iss 2, Pp 182-191 (2014), Antibodies; Volume 3; Issue 2; Pages: 182-191
Publication Year :
2014
Publisher :
MDPI AG, 2014.

Abstract

We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag) is tightly bound by a commercial antibody when expressed at the C-term end of a polypeptide. The antigen is released in the presence of 2 M MgCl2. Consequently, constructs fused to the 6xHis-C tags can be purified by two successive and orthogonal affinity steps. Single-domain antibodies were produced either in the periplasmic or in the cytoplasmic space of E. coli. Surprisingly, the first affinity purification step performed using the EPEA-binding resin already yielded homogeneous proteins. The presence of the C tag did not interfere with the binding activity of the antibodies, as assessed by FACS and SPR analyses, and the C tag was extremely effective for immunoprecipitating HER2 receptor. Finally, the Alexa488-coupled anti-C tag allowed for simplification of FACS and IF analyses. These results show that a tag of minimal dimensions can be effectively used to improve the applicability of recombinant antibodies as reagents. In our hands, C tag was superior to His-tag in affinity purification and pull-down experiments, and practical in any other standard immune technique.

Details

ISSN :
20734468
Volume :
3
Database :
OpenAIRE
Journal :
Antibodies
Accession number :
edsair.doi.dedup.....ee0d3ccdf3c1cd25783796dd466b0041