EXPRESSION OF LIGNIN PEROXIDASE H8 IN ESCHERICHIA-COLI - FOLDING AND ACTIVATION OF THE RECOMBINANT ENZYME WITH CA2+ AND HEME

An engineered cDNA from Phanerochaete chrysosporium encoding both the mature and pro-sequence regions of Lip isoenzyme H8 (Lip) has been suc cessfully overexpressed in Escherichia coli. The recombinant protein ( LipP) was sequestered in inclusion bodies. The reduced-denatured poly peptide has been purified by differential solubilization, and the acti ve enzyme recovered after controlled in vitro refolding (albeit in low yield), by glutathione-mediated oxidation of disulphides, in a foldin g medium containing an intermediate concentration of urea, Ca2+ and ha em. The procedure is analogous to that previously described for the pr oduction of active recombinant horseradish peroxidase (HRP-C) from in clusion-body material. It is quite possible, therefore, that this type of procedure may be suitable for the recovery of most, if not all, ac tive recombinant peroxidases. The resultant LipP has spectral charact eristics identical with that of the native enzyme as isolated from Pha nerochaete chrysosporium. Its specific activity measured in the standa rd veratryl alcohol (VA) assay was 39 mu mol of VA oxidized/min per mg of protein, a value which compares extremely favourably with that of the native enzyme (36 mu mol of VA/min per mg). Although levels of act ive enzyme obtained are not yet as high as in the case of HRP-C (1% c onversion of crude inactive LipP polypeptide into pure fully active L ip), it is envisaged that further refinement of the expression/folding /activation procedures will provide sufficient protein for biophysical characterization of both the wild-type and site-directed mutants.