Functional expression of recombinant anti-BNP scFv in methylotrophic yeast Pichia pastoris and application as a recognition molecule in electrochemical sensors.
Recent studies have revealed the potential of B-type natriuretic peptide (BNP) as a good prognostic marker for patients with heart failure. Antibodies against BNP are expected to be usefully employed in the diagnosis of heart failures. We established a more efficient method to produce functional anti-BNP, single chain variable fragment (scFv) using a eukaryotic expression system of Pichia pastoris. Although analysis of the N-terminal (NT) sequence of the expressed anti-BNP scFv indicated that the two Ste13 sites of the secreted anti-BNP scFv were not cleaved, the specificity of anti-BNP scFv was not affected significantly. The binding activity of anti-BNP scFv against other antigens, against four other antigens, NT probrain peptide (NT-pro BNP), atrial natriuretic peptide (ANP), carcinoembryonic antigen (CEA) and human serum albumin (HSA), was only one thousandth that of the original BNP antigen, which clearly demonstrated the specific binding of recombinant scFv toward BNP. The anti-BNP, scFv-based, electrochemical immunoassay exhibited excellent analytical performance with a detection limit of 1 fg/ml and a wide linear detection range from 1 to 10,000 fg/ml. The optimum culture conditions to obtain the maximum concentration of recombinant scFv were a pH range of 5.0-7.0 and an incubation temperature of 20°C. This anti-BNP scFv expressed in P. pastoris has the potential for promising applications in the diagnosis of heart failure.
Department of Chemical Engineering, Kwangwoon University, 447-1 Wolgye-dong, Nowon-gu, Seoul 139-701, Republic of Korea.
Bo Hee
BH
Maeng
Jeongyeon
J
Choi
Young Seung
YS
Sa
Jae Ho
JH
Shin
Yong Hwan
YH
Kim
D019411:Clinical Laboratory Techniques / Q000379:methods*; D055664:Electrochemical Techniques / Q000379:methods*; D015870:Gene Expression; D006333:Heart Failure / Q000175:diagnosis*; D006801:Humans; D007118:Immunoassay; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020097:Natriuretic Peptide, Brain / Q000097:blood*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity
D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D020097:Natriuretic Peptide, Brain