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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)
- 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.
- Subjects :
- lcsh:Immunologic diseases. Allergy
0106 biological sciences
Immunology
VHH
Protein tag
Biology
immunoprecipitation
01 natural sciences
antibody screening
tandem affinity purification
law.invention
03 medical and health sciences
Affinity chromatography
Antigen
law
010608 biotechnology
Drug Discovery
Immunology and Allergy
Peptide sequence
cytoplasmic antibody expression
nanobody
030304 developmental biology
Tandem affinity purification
0303 health sciences
Periplasmic space
Molecular biology
Recombinant DNA
lcsh:RC581-607
Myc-tag
Subjects
Details
- ISSN :
- 20734468
- Volume :
- 3
- Database :
- OpenAIRE
- Journal :
- Antibodies
- Accession number :
- edsair.doi.dedup.....ee0d3ccdf3c1cd25783796dd466b0041