Expression, purification, and characterization of equistatin in Pichia pastoris

Equistatin (EI) is a cysteine protease inhibitor that was isolated from the sea anemone Actinia equina It belongs to a recently discovered group of th yroglobulin type-I domain inhibitors called thyropins. Since native EI is f ound only in low amounts in the body of sea anemone and expression of recom binant EI in Escherichia coli yielded only 1 mg/liter of protein, we used t he Pichia pastoris expression system to obtain higher yields. A cDNA encodi ng EI was inserted into pPIC9 vector and transformed into the P, pastoris, strain GS115. Clones expressing high levels of EI were selected from 48 tra nsformants. Recombinant EI was produced in 2-liter shake flasks and recover ed from the fermentation broth by affinity chromatography using CM-papain-S epharose. SDS-PAGE and N-terminal sequence analysis revealed that EI was N- terminally intact and running at; the expected molecular weight of 22 kDa. The equilibrium dissociation constants of EI with papain and bovine catheps in D were determined and were found to be similar to the results for the na tive inhibitor. EI production was scaled up to a bench top fermenter with a 25 mg/liter yield of active EI, (C) 2000 Academic Press.