A single chain antibody (scFvNC10.14) that binds the superpotent sweetener ligand N-(p-cy an ophen y 1 )-N′- (diphenylmethyl)guanidine acetic acid was produced in a recombinant bacterial expression system. ScFvNC10.14 was constructed with isolated total mRNA from IgG2b, \producing hybridoma cells, using reverse transcriptase and PCR to amplify the cDNA coding for the V and J regions of the L and H chains. The cDNA fragments were cloned into a pre-existing vector with a 26 amino acid linker. Additional PCR incorporated a hexahistidine tag to the C-terminus of the protein, and the resulting cDNA was cloned into pET-lla for expression. ScFvNClO. 14 was purified from inclusion bodies under denaturing conditions using N12+-NTA agarose, and refolded by stepwise dialysis in decreasing concentrations of urea, with a final dialysis in phosphate buffer. Far UV circular dichroism (CD) revealed that the secondary structure of scFvNC10.14 possessed predominant β-character. Near UV CD showed chromophore perturbation occurred upon ligand binding, and quenching of aromatic residue fluorescence was observed by fluorescence spectroscopy. The Kd for ligand binding of scFv was determined by RIA to be 20 nM (18 nM for whole leG). The properties of scFvNC10.14 appear to be nearly identical to that of the parent IgG.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Molecular Biology