title: Expression and purification of a recombinant metal-binding T4 lysozyme fusion protein.
abstract: Periplasmic expression of recombinant proteins presents many potential benefits that may aid recovery of the protein product. Muramidases are the preferred agents in effecting selective release of recombinant proteins from the periplasm of E. coli and other Gram negative bacteria. Unfortunately cost restricts the use of pure lytic enzymes at large-scale and their removal as process contaminants adds to later purification demands. We constructed a reusable version of bacteriophage T4 lysozyme, by fusing a His-Gln-(His)3 peptide sequence to the C-terminus of a cysteine-free pseudo wild type bacteriophage T4 lysozyme. The peptide tail allowed rapid and high-level recovery on IDA Sepharose columns charged with Zn2+, Ni2+ and Cu2+ ions. The binding to metal-charged supports was specifically mediated by the histidine-rich tail as no binding was observed for the original cysteine-free pseudo wild type lysozyme. The strength of retention of polyhistidine recombinant T4 lysozyme on charged supports followed the expected Cu > Ni > Zn pattern, but there were few differences in the levels of purity and recovery of the modified enzyme, from columns charged with the different metal ions.
authors.affiliation: Advanced Centre for Biochemical Engineering, University College London, UK.
authors.forename: R P
authors.initials: RP
authors.lastname: Sloane
authors.forename: J M
authors.initials: JM
authors.lastname: Ward
authors.forename: S M
authors.initials: SM
authors.lastname: O'Brien
authors.forename: O R
authors.initials: OR
authors.lastname: Thomas
authors.forename: P
authors.initials: P
authors.lastname: Dunnill
mesh_terms: D003545:Cysteine / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008670:Metals / Q000378:metabolism; D009113:Muramidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D008670:Metals; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C094995:T4 lysozyme (38-51); D006639:Histidine; D009113:Muramidase; D003545:Cysteine