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

Antibodies

Selma Djender¹  Anne Beugnet¹  Aurelie Schneider¹ 

[1] Tumor target and therapeutic antibody-Identification Platform (TAb-IP), Institut Curie, 3-5 Impasse Reille, 75014 Paris, France

关键词: antibody screening;    cytoplasmic antibody expression;    immunoprecipitation;    nanobody;    VHH;    tandem affinity purification;   

DOI  :  10.3390/antib3020182

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.