Characterisation of non-porous magnetic chelator supports and their use to recover polyhistidine-tailed T4 lysozyme from a crude E. coli extract.
The use of high capacity micron-sized non-porous magnetic metal chelator adsorbents for the direct recovery of a recombinant metal-binding protein from crude liquors is described. Selectivity and interaction strength of magnetic chelator particles were assessed using a set of native proteins with known behaviour towards commercially available immobilised metal chelate adsorbents. Particles charged with Cu2+ were highly effective in recovering a recombinant histidine-tailed T4 lysozyme fusion protein directly from crude E. coli extracts in a single step. Levels of recovery and purity were high and compared favourably with those achieved by chromatography of pre-clarified extracts on Cu(2+)-IDA Sepharose. The magnetic approach offers advantages such as the avoidance of clarification to prevent fouling of chromatography columns, steps that become especially significant at large scale. By detailed characterisation of the magnetic chelators the practical use of tailed T4 lysozyme for repeated production of periplasmic products is a realistic prospect.
O'Brien
S M
SM
Department of Chemical and Biochemical Engineering, University College London, UK.
Sloane
R P
RP
Thomas
O R
OR
Dunnill
P
P
D017122:Bacteriophage T4 / Q000201:enzymology*; D019149:Bioreactors; D002614:Chelating Agents / Q000378:metabolism*; D002846:Chromatography, Affinity; D003300:Copper / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine; D008280:Magnetics; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010455:Peptides / Q000032:analysis; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose
D002614:Chelating Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D003300:Copper; D012685:Sepharose; D009113:Muramidase