Sanya J. Sanderson

Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture.

Functional conservation of a natural cysteine peptidase inhibitor in protozoan and bacterial pathogens

Cysteine peptidase inhibitor genes (ICP) of the chagasin family have been identified in protozoan (Leishmania mexicana and Trypanosoma brucei) and bacterial (Pseudomonas aeruginosa) pathogens. The encoded proteins have low sequence identities with each other and no significant identity with cystatins or other known cysteine peptidase inhibitors. Recombinant forms of each ICP inhibit protozoan and mammalian clan CA, family C1 cysteine peptidases but do not inhibit the clan CD cysteine peptidase caspase 3, the serine peptidase trypsin or the aspartic peptidases pepsin and thrombin. The functional homology between ICPs implies a common evolutionary origin for these bacterial and protozoal proteins.

The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: application of a combinatorial peptide library approach.

The substrate specificity of CPB2.8ΔCTE, a recombinant cysteine protease from Leishmania mexicana, was mapped by screening a fluorescence‐quenched combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S3 subsite and for hydrophobic residues, both aliphatic and aromatic, in S2. The S1 subsite exhibited a specificity for the basic residues Arg and Lys. Generally, the specificity of the primed subsites was less strict compared with the non‐primed side which showed preference for Arg, Lys and Ala in S

Analysis of the S 2 subsite specificities of the recombinant cysteine proteinases CPB of Leishmania mexicana, and cruzain of Trypanosoma cruzi, using fluorescent substrates containing non-natural basic amino acids

_{1}^{\prime }

Identification of peptides inhibitory to recombinant cysteine proteinase, CPB, of Leishmania mexicana.

, Arg, Pro and Gly in S

A combinatorial approach to the identification of cysteine protease substrates and inhibitors by application of a solid-phase fluorescence quenching assay

_{2}^{\prime }

Combinatorial library of peptidotriazoles: identification of [1,2,3]-triazole inhibitors against a recombinant Leishmania mexicana cysteine protease.

and Lys, Arg and Ser in S

Stoichiometry, organisation and catalytic function of protein X of the pyruvate dehydrogenase complex from bovine heart

_{4}^{\prime }

Reconstitution of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes: analysis of protein X involvement and interaction of homologous and heterologous dihydrolipoamide dehydrogenases.

. By contrast, a strict preference for the basic residues Arg and Lys was found for S

Solid-Phase Library Synthesis, Screening, and Selection of Tight-Binding Reduced Peptide Bond Inhibitors of a Recombinant Leishmania mexicana Cysteine Protease B

_{3}^{\prime }