Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20.

摘要: Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition the relative roles three light chain antigen (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32phe, L34arg L34lys L34his) were generated expressed single-chain binding derivatives containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids SCA 4-4-20/212, 14 acids). Results showed that L27dhis necessary for wild type affinities, however, not required near-wild Qmax values (where is maximum fluoroscein fluorescence quenching as percent). Tyrosine L32 which hydrogen bonds was also characterized at haptenic level through use 9-hydroxyphenylfluoron lacks carboxyl group tyrosine forms a bond. demonstrated mutant L32phe possessed similar HPF characteristics. Active residue important fluorescein maxima (L34his mutant), substitution lysine arginine L34 did have significant effect on observed value. In addition, substitutions had no structural topological characteristics, since all mutants retained idiotypic metatypic properties. Finally, comparatively examined determine contributions properties stability. No linker effects observed. Collectively, these results verified importance pocket 4-4-20.