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Antibodies 2014, 3(2), 182-191; doi:10.3390/antib3020182
Article

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

1, 1, 1 and 1,2,3,4,*
1 Tumor target and therapeutic antibody-Identification Platform (TAb-IP), Institut Curie, 3-5 Impasse Reille, 75014 Paris, France 2 SIRIC INCa-DGOS-4654, Institut Curie, F-75248 Paris Cedex 05, France 3 CIC IGR Curie 1428, Institut Curie, F-75248 Paris Cedex 05, France 4 Dept. of Biomedical Sciences and Engineering, University of Nova Gorica (UNG), Glavni Trg 9-SI-5261, Vipava, Slovenia
* Author to whom correspondence should be addressed.
Received: 11 February 2014 / Revised: 18 March 2014 / Accepted: 20 March 2014 / Published: 2 April 2014
(This article belongs to the Special Issue Antibody Constructs)
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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.
Keywords: antibody screening; cytoplasmic antibody expression; immunoprecipitation; nanobody; VHH; tandem affinity purification antibody screening; cytoplasmic antibody expression; immunoprecipitation; nanobody; VHH; tandem affinity purification
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Djender, S.; Beugnet, A.; Schneider, A.; de Marco, A. The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag). Antibodies 2014, 3, 182-191.

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