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,* email
Received: 11 February 2014; in revised form: 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
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

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.

AMA Style

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(2):182-191.

Chicago/Turabian Style

Djender, Selma; Beugnet, Anne; Schneider, Aurelie; de Marco, Ario. 2014. "The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag)." Antibodies 3, no. 2: 182-191.

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