Immobilised metal affinity chromatography of beta-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption.
The development of an expanded bed process for the direct extraction and partial purification of beta-galactosidase from unclarified Escherichia coli homogenates using its natural affinity for metal loaded STREAMLINE Chelating is described. Small packed beds were used to determine the effect of chelated metal ion (Cu2+, Ni2+, Co2+ or Zn2+), loading pH and ionic strength on the selective binding capacity, and recovery of beta-galactosidase from clarified homogenates. An elution protocol was developed using the competitive displacer, imidazole, to recover beta-galactosidase in 87% yield and 3.4-fold purification. These results were then used to develop a separation for the recovery of beta-galactosidase from unclarified homogenates in a 2.5-cm diameter expanded bed. Although Ni2+ loaded STREAMLINE Chelating had a 5% dynamic capacity for beta-galactosidase of just 118 U ml(-1) (0.39 mg ml(-1)), the low capacity was thought to be due to the large size of the target (464,000) relative to the exclusion limit of the macroporous adsorbent. Despite this low capacity, Ni2 STREAMLINE Chelating was used successfully to recover beta-galactosidase from an unclarified homogenate in 86.4% yield and at 5.95-fold purification. The degree of purification relative to a commercial standard, as assessed using the purification factor and sodium dodecyl sulphate-polyacrylamide gel electrophoresis was high suggesting that this pseudo-affinity procedure compared favourably with alternative methods.
Clemmitt
R H
RH
Department of Chemical Engineering, University of Cambridge, UK.
Chase
H A
HA
D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D009994:Osmolar Concentration; D001616:beta-Galactosidase / Q000302:isolation & purification*
D002614:Chelating Agents; D007093:Imidazoles; D008670:Metals; C029899:imidazole; D009532:Nickel; D001616:beta-Galactosidase