Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10843337 | Protein Expression and Purification | 2010 | 7 Pages |
Abstract
Single-domain single-chain variable fragment (scFv) antibody is sometimes critical for purification using affinity tagging strategy. We failed in our initial effort to purify a prematurely developed Camelid-like E-tagged short scFv-K2 antibody that contained a complete variable region of the heavy chain and partial region of the light chain by using an anti-E-tag affinity column. To expedite the purification of this altered but interesting antimycotic agent, we replaced a long and large E-tag by a short and hydrophilic 6Ã-Histidine (His6) affinity tag by polymerase chain reaction. The short and compact His6-tag was placed on the previously constructed expression vector pCANTAB 5 E that contained the large affinity E-tag sequence (13 amino acids) by PCR-based mutagenesis and was expressed in Escherichia coli. The recombinant protein can then be purified by immobilized metal affinity chromatography (IMAC) and be used for biochemical and other functional characterization. This His6-tagged short scFv-K2 antibody (20Â kDa) had strong cytocidal activity against Saccharomyces and Candida species with a IC50 value of 0.44Â ÃÂ 10â6Â M and 1.10 Ã 10â6Â M, respectively. Tag replacement facilitates the purification of a Camelid-like single-domain scFv antibody and after that meets its different functional characteristics. The present study reflects that the VH domain of the scFv antibody is mainly responsible for its biological activity and single-domain scFv antibody may acts as a potent antimicrobial agent.
Keywords
CDRIPTGscFvIMACHis6variable region of the light chainSDS–PAGEsodium dodecyl sulfate–polyacrylamide gel electrophoresisisopropyl β-D-1-thiogalactopyranosideELISAEnzyme-linked immunosorbent assaysingle-chain variable fragmentcomplementarity determining regionpolymerase chain reactionPCRimmobilized metal affinity chromatography
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Authors
M. Enamul Kabir, Senthilkumar Krishnaswamy, Masahiko Miyamoto, Yasuhiro Furuichi, Tadazumi Komiyama,