A genetic approach to the generation of antibodies with enhanced catalytic activities.
A hydrolytic catalytic antibody, generated against a nitrophenyl phosphonate transition state analogue, has been cloned and expressed in Escherichia coli for use as a model system to demonstrate the feasibility of using genetic selections to enhance catalytic activity. Conditions were found that permit the secretion of active recombinant antibody into the periplasm of a strain of E. coli deficient in the biotin biosynthetic genes (delta bio-gal). A number of substrates were synthesized that, upon hydrolysis by the antibody, yield free biotin, which is required for cell growth. The substrates and selections can be used to identify mutants of the antibody with altered activities. This approach should be generalizable to a wide number of hydrolytic reactions including the selective cleavage of peptide, polysaccharide, phosphodiester, and ester bonds.
Lesley
S A
SA
Department of Chemistry, University of California, Berkeley 94720.
Patten
P A
PA
Schultz
P G
PG
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006241:Haptens; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A