Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system.

A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN-III-ompA Escherichia coli expression system. After osmotic shock of the E. coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S-carboxymethylpapain affinity chromatography. The resulting inhibitory material was characterized by SDS/PAGE, reversed-phase HPLC, peptide mapping and amino acid sequencing. The recombinant variant chicken AEF-[S1----M, M29----I, M89----L]cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin. The purified variant showed a native-chicken-cystatin-like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N-labelled recombinant inhibitor were compared with those of the natural inhibitor.