Construction, bacterial expression, and characterization of hapten-specific single-chain Fv and alkaline phosphatase fusion protein.
We have designed and constructed a bacterial expression vector to produce a fusion protein of hapten-specific single-chain Fv (ScFv) and alkaline phosphatase (PhoA) in Escherichia coli. The ScFv gene was assembled using genes encoding the heavy and light chain variable domains of anti-NP (4-hydroxy-3-nitrophenyl acetyl) mouse monoclonal antibody. The ScFv gene was then fused to the 5' terminus of the E. coli PhoA coding region. The expressed fusion protein ScFv(NP)-PhoA was purified using an NP affinity column, and gel-filtration. Characterization of the fusion protein was then performed. The estimated molecular weight by gel filtration was approximately 151 kDa, suggesting the dimerization of the protein. Kinetic constants of ScFv(NP)-PhoA were calculated and compared with those of wild-type PhoA. The k(cat) values of ScFv(NP)-PhoA and wild-type PhoA were 103 (s(-1)) and 96.1 (s(-1)), respectively, showing that PhoA activity was somewhat increased by tethering the molecules. The equilibrium binding constant of ScFv(NP)-PhoA was determined using two different haptens, NP-capronate and NIP(3-iodo-4-hydroxy-5-nitrophenyl acetyl) by means of fluorescence quenching measurements. The obtained binding constants were 2.2 x 10(5) (M-1) for NP-capronate and 1.O x 10(6) (M(-1)) for NIP, respectively. No apparent difference in binding constants was seen between ScFv(NP) and ScFv(NP)-PhoA, showing that sufficient specificity and binding affinity were retained when ScFv(NP) was tethered to alkaline phosphatase. ScFv(NP)-PhoA can be used to detect nanogram concentrations of NP-BSA in ELISA without the use of chemically conjugated secondary antibodies.
Suzuki
C
C
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo. chikakos@bio.t.u-tokyo.ac.jp
Ueda
H
H
Suzuki
E
E
Nagamune
T
T
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006241:Haptens / Q000032:analysis; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009596:Nitrophenols / Q000032:analysis; D010455:Peptides / Q000235:genetics; D010648:Phenylacetates; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006241:Haptens; D007128:Immunoglobulin Fragments; D009596:Nitrophenols; D010455:Peptides; D010648:Phenylacetates; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; C026242:4-hydroxy-5-nitrophenyl acetic acid; D000469:Alkaline Phosphatase