Purification and functional characterization of a Camelid-like single-domain antimycotic antibody by engineering in affinity tag.
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 6x-Histidine (His(6)) affinity tag by polymerase chain reaction. The short and compact His(6)-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 His(6)-tagged short scFv-K2 antibody (20 kDa) had strong cytocidal activity against Saccharomyces and Candida species with a IC(50) value of 0.44x10(-6)M and 1.10 x 10(-6)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 V(H) domain of the scFv antibody is mainly responsible for its biological activity and single-domain scFv antibody may acts as a potent antimicrobial agent.
Kabir
M Enamul
ME
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata 956-8603, Japan.
Krishnaswamy
Senthilkumar
S
Miyamoto
Masahiko
M
Furuichi
Yasuhiro
Y
Komiyama
Tadazumi
T
D000818:Animals; D000908:Antibodies, Fungal / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D000935:Antifungal Agents / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D015153:Blotting, Western; D002175:Candida / Q000187:drug effects; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology*; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D012418:Ruminants; D012440:Saccharomyces / Q000187:drug effects
D000908:Antibodies, Fungal; D000935:Antifungal Agents; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins