Cloning and expression of functional equistatin

Equistatin is a 199-residue protein composed of three thyroglobulin type-1 domains. It strongly inhibits cysteine proteinases as well as the aspartic proteinase cathepsin D. In order to initiate structure-function studies by protein engineering, a cDNA library from sea anemone, Actinia equina, was s creened. A positive clone of 888 nucleotides was shown to encode a protein of 231 amino acids, including the signal sequence. The mature protein regio n was amplified by PCR, cloned into the pET22b(+)cas expression vector and expressed in Escherichia coli. Isolation of active recombinant equistatin r equired only one purification step, the His-tag affinity column. The protei n displays physical and inhibitory properties closely similar to the native inhibitor.