[Optimized expression of a single-chain Fv antibody against human asialoglycoprotein receptor and determination of its affinity constant].
OBJECTIVE
To express and purify a single chain Fv antibody (scFv) C1 against human hepatic asialoglycoprotein receptor (ASGPR) and to determine affinity constant of the purified scFv C1.


METHODS
The specific anti-ASGPR phage clone C1 was transfected into E. coli HB2151. The single colony was chosen to be inoculated into 2 x TY medium and shaken (250 r/min) overnight at 37 degrees C. After 1 in 100 dilution in 2 x TY medium and induced for secreted expression, scFv C1 was induced at different concentrations (0.25, 0.5 and 1.0 mmol/L IPTG) overnight at 37 degrees C, 25 degrees C and 20 degrees C, respectively. The supernatant was precipitated with saturated ammonium sulfate and its sediment was analyzed by SDS-PAGE. In addition, the sediment was resuspended in 30 mL PBS and dialyzed against PBS overnight at 4 degrees C. The expressed scFv C1 was purified by Ni(2+) chelating HiTrap HP column and the purity of the purified scFv C1 was identified by SDS-PAGE. Then affinity constant of scFv C1 was determined by noncompetitive enzyme immunoassay.


RESULTS
After induced with 0.5 mmol/L IPTG overnight at 25 degrees C, the amount of expressed scFv C1 increased greatly and its relative molecular mass was about 28,000, and it existed in culture supernant in soluble form. The purity of scFv C1 by nickel-agarose column was above 95% and its yield was about 0.8 mg/L. The affinity constant of the purified scFv C1 was confirmed to be (2.31+/-0.36)x10(-7) mol/L.


CONCLUSIONS
The E. coli HB2151 infected with phage C1 clones may express soluble scFv C1 with low affinity, which has potential applications to gene therapy of hepatocellular carcinoma.
Wang
Wei-yu
WY
Department of General Surgery, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. wwytj2000@163.com
Yi
Ji-lin
JL
Deng
Yun-hua
YH
Si
Jin
J
Wang
Cong-jun
CJ
Li
Xiang
X
Cao
Li-min
LM
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D000915:Antibody Affinity; D037263:Asialoglycoprotein Receptor / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015316:Genetic Therapy; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D008113:Liver Neoplasms / Q000235:genetics / Q000628:therapy; D013696:Temperature
D000911:Antibodies, Monoclonal; D037263:Asialoglycoprotein Receptor; D007135:Immunoglobulin Variable Region