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

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