Probing the importance of second sphere residues in an esterolytic antibody by phage display.
We have used phage display to generate a panel of closely related catalytic antibodies. Seeking to improve the catalytic activity of an esterolytic antibody, we displayed libraries derived from the humanized Fab fragment of the antibody 17E8 (h17E8) on filamentous phage and sorted for binding to an immobilized transition-state analog (TSA). Previous work had suggested that residues outside the antibody active site contribute to TSA binding and catalytic efficiency, and we tested this notion by generating libraries containing such "second sphere" residues. Selected variants of h17E8 retained esterolytic activity and showed variations in affinity within 40-fold and kinetic parameters within tenfold of wild-type antibody, indicating that residues remote from the active site do modulate catalytic activity. In order to understand which mutations were responsible for the properties of phage-selected variants, we designed a series of site-directed mutants. From this series, we identified a double mutant in which Tyr97 was changed to Arg in the heavy chain (Y97HR) and the heavy chain Tyr100a was mutated to Asn (Y100aHN). This variant showed a tenfold improvement in catalytic efficiency (kcat/KM) relative to wild-type h17E8. These mutations were additive; Y97HR increases the catalytic turnover (kcat) by three- to fourfold, while Y100aHN has been shown to lower the Michaelis constant (KM) by three- to fivefold. TSA binding was correlated with catalytic turnover for variants that differed by single mutations, but less so for variants that differed by many mutations. Thus, future selections based on TSA binding should focus on mutating a small number of residues at a time.
Arkin
M R
MR
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, 94080, USA.
Wells
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D004247:DNA / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation
D017151:Antibodies, Catalytic; D007135:Immunoglobulin Variable Region; D004247:DNA