RAT THIMET OLIGOPEPTIDASE - LARGE-SCALE EXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

The coding sequence for rat testis thimet oligopeptidase (TOP) (EC 3.4 .24.15) was placed under the control of the T7 polymerase/promoter sys tem. Cultures of Escherichia coli transfected with the resulting plasm id expressed the enzyme as a soluble cytoplasmic protein. Medium-scale cultures allowed isolation of the enzyme in quantities of tens of mil ligrams. The availability of the recombinant enzyme permitted the dete rmination of such chemical properties as epsilon(280) (48960), zinc co ntent (2 atom/molecule) and available thiol content (8-10/molecule) fo r TOP. The recombinant enzyme showed the catalytic activities previous ly reported for the naturally occurring enzyme, so that we can now con clude with confidence that these are all due to TOP and there is no ne ed to postulate the existence of separate 'Pz-peptidase' or 'endo-olig opeptidase A' enzymes.