EXPRESSION AND PURIFICATION OF A RECOMBINANT METAL-BINDING T4 LYSOZYME FUSION PROTEIN

Periplasmic expression of recombinant proteins presents many potential benefits that may aid recovery of the protein product. Muramidases ar e the preferred agents in effecting selective release of recombinant p roteins from the periplasm of E. coli and other Gram negative bacteria . Unfortunately cost restricts the use of pure lytic enzymes al large- scale and their removal as process contaminants adds to later purifica tion demands. We constructed a reusable version of bacteriophage T4 ly sozyme, by fusing a His-Gln-(His)(3) peptide sequence to the C-terminu s of a cysteine-free pseudo wild type bacteriophage T4 lysozyme. The p eptide tail allowed rapid and high-level recovery on IDA Sepharose col umns charged with Zn2+, Ni2+ and Cu2+ ions. The binding to metal-charg ed 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 ly sozyme on charged supports followed the expected Cu > Ni > Zn pattern, but there were few differences in the levels of purity and recovery o f the modified enzyme, from columns charged with the different metal i ons.