Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: antibodies for orotate decarboxylation.
Antibodies capable of decarboxylating orotate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate-binding and catalytic portion of the active site of the enzyme orotidine 5'-monophosphate (OMP) decarboxylase (orotidine-5'-monophosphate carboxy-lyase, EC 4.1.1.23). Active recombinant antibody fragments (Fabs) were selected from a combinatorial cDNA library by complementation of a pyrF strain of Escherichia coli and growth of the library-expressing cells on pyrimidine-free medium. In this biological screen, a sufficiently active antibody from the library would decarboxylate orotate to produce uracil, a pyrimidine source for the auxotroph, and would provide the cells with a growth advantage compared to cells without an active antibody. Six recombinant Fabs yielded identifiable colonies in a screen of 16,000 transformants. To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form. In this form, the antibody fragment conferred a definite growth advantage to the auxotroph that was eliminated when the hapten was included in the medium. The purified single-chain antibody displayed orotate decarboxylase activity in vitro, as determined by a 14CO2 displacement assay. The specific activity of the antibody is approximately 10(-7) times that of naturally occurring OMP decarboxylase, but this antibody-catalyzed rate is estimated to be 10(8) times the background rate. The results offer the potential to use these methods to obtain catalytic antibodies for other biosynthetic reactions as well as to assess the effectiveness of the hapten transition state or active site analog in eliciting antibody catalysts.
Smiley
J A
JA
Department of Chemistry, Pennsylvania State University, University Park 16802.
Benkovic
S J
SJ
D017151:Antibodies, Catalytic; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005816:Genetic Complementation Test; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D009963:Orotic Acid / Q000378:metabolism*; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000378:metabolism; D011994:Recombinant Proteins
D017151:Antibodies, Catalytic; D018076:DNA, Complementary; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D009963:Orotic Acid; D009964:Orotidine-5'-Phosphate Decarboxylase