Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds

Two cysteine proteinase inhibitors, cystatins Sea and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J, Biochem, 119 (1996) 1106-1 113]. A cDNA clone encoding a novel phytocystatin with three repetitive cys tatin domains was isolated from a cDNA library of sunflower seeds using the Sea cDNA fragment as a hybridization probe. The cDNA insert comprises 1,09 3 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% id entical residues with Sea, The cDNA was expressed in Escherichia coli cells , and then the recombinant sunflower multicystatin (SMC) was purified and i ts inhibitory activity toward papain was examined. SMC exhibited strong inh ibitory activity toward papain, with a stoichiometry of 1:3, indicating tha t each cystatin domain independently functions as a potent cysteine protein ase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested th at post-translational processing by an Asn-specific proteinase may give ris e to mature Sca-like phytocystatins.