The Purification of β-Galactosidase from Escherichia coli by Affinity Chromatography

Abstract The chromatographic behavior of β-galactosidase from the constitutive strain 3300 of Escherichia coli, K-12, was studied with derivatives of agarose and polyacrylamide which contained the substrate analogue inhibitor, p-aminophenyl-β-dthiogalactopyranoside, covalently attached in various ways. A two-step purification of this enzyme from extracts could be achieved with selective agarose adsorbents provided the ligand was placed at a sufficient distance (about 21 A) from the matrix backbone by interposing a long hydrocarbon arm, 3-aminosuccinyl-3'-aminodipropylamine. The various polyacrylamide-ligand derivatives studied were ineffective in adsorbing the enzyme. The enzyme adsorbed tightly at neutral pH to columns containing the substituted resin, and elution was achieved with buffers having a pH of 10. Buffers containing high concentration (0.05 m) of the substrates, lactose, isopropyl-β-d-galactopyranoside, or o-nitrophenyl-β-d-galactopyranoside, did not affect elution of the enzyme and were rapidly hydrolyzed by the enzyme adsorbed to the column. The enzyme obtained by these procedures was pure by disc gel electrophoresis and it possessed the physical and chemical characteristics of the protein purified by conventional procedures. The specific activity was between 300,000 and 320,000 units per mg. The procedure can be carried out on a preparative scale, and the adsorbent may be used several times without appreciable impairment of the binding capacity. The enzyme remains active while bound to the column and it can be readsorbed and eluted without apparent deleterious effects. The monomeric form of the enzyme also binds strongly to the substituted agarose, and it can be eluted together with the active tetramer species with sodium borate, pH 10. The ability of the enzymatically inactive monomer forms to bind to the ligand column suggests that this affinity chromatographic method may be used in the purification of catalytically inactive mutant forms which retain the ability to bind substrate analogues.