Complementable Fraction and Complemented Enzyme of Mutant Ml5 from Escherichia Coli: Partial Purification by Affinity Chromatography

Abstract

The technique of affinity chromatography has been used in the partial purification of complementable fractions and complemented enzyme of β-galactosidase from Escherichia coli mutant M15. The crude extract of mutant ML5 was incubated with fragment CM-B. The complemented enzyme and complementable fractions were passed through a small column of p-amino-phenyl-β-D-thiogalactoside to which inhibitors had been covalently attached. A high percentage of the nonspecific protein passed directly through the affinity column while the specific enzymatic protein remained bound to the gel. Phosphate buffer with NaCl was used to elute the complementable fractions from the column. Sodium borate buffer was used to elute the bound complemented enzyme from the affinity support. The results of this study show that 100% of the complemented enzyme was bound to the column. The partially purified enzyme had the same position in disc gel electrophoresis as β-galactosidase from E. coli.