Affinity chromatography on anti-B1 monoclonal gels for purification and characterization of protein B1 from Escherichia coli ribonucleotide reductase.
Affinity gels were prepared from four monoclonal antibodies against the B1 protein of ribonucleotide reductase of Escherichia coli. The gels were used to purify protein B1 and also to study some of its properties. Gels from the nonneutralizing monoclonal anti-B1-k bound as much as 2 mg of B1/mL and were employed to prepare essentially pure B1 protein in a single step from extracts of wild-type E. coli and strains overproducing the subunit. However, B1 prepared from wild-type extracts had a lowered specific activity, suggesting some denaturation during elution of the protein from the column. Addition of the allosteric effector dATP during affinity chromatography changed the chromatographic pattern. Some protein B2, the second subunit of the reductase, remained in all cases bound to the gels together with B1. The gel prepared from anti-B1-c retained two additional proteins. In other experiments involving binding of proteolytic fragments of B1 to various antibodies, we also found a striking effect of dATP, suggesting that dATP made protein B1 less accessible to proteolysis. In these experiments fragments around 15K still had the ability to bind monoclonals, making possible more detailed investigations of the structural contacts between B1 and the monoclonals.
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D003838:Deoxyadenine Nucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D012264:Ribonucleotide Reductases / Q000032:analysis* / Q000276:immunology; D013381:Subtilisins; D014357:Trypsin
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003838:Deoxyadenine Nucleotides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012264:Ribonucleotide Reductases; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin; C026600:2'-deoxyadenosine triphosphate