Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predomina ntly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodi es to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an ina ctive pro-form lacking the characteristic C-terminal extension (CPB2.8 Delt a CTE). Pro-region processing was initiated during protein refolding and pr oceeded through several intermediate stages. Maximum enzyme activity accomp anied removal of the entire pro-region. This was facilitated by acidificati on. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/P AGE gels, co-migrated with native enzyme in L. mexicana lysates, and had th e same N-terminal sequence as the native enzyme. The procedure yielded > 3. 5 mg of active enzyme per litre of E. coli culture.