A RECOMBINANT WHEAT SERPIN WITH INHIBITORY ACTIVITY

A full-length clone encoding the wheat (Triticum aestivum L.) serpin W SZ1 was isolated from a cDNA library based on mRNA from immature grain . The 398 amino acid sequence deduced from the cDNA was corroborated b y sequencing CNBr peptides of WSZ1 purified from resting grain. WSZ1 b elongs to the subfamily of protein Z-type serpins and the amino acid s equence is 70%, identical with the barley serpins BSZ4 and BSZx and 27 -33% identical with human serpins such as alpha(1)-proteinase inhibito r, antithrombin III, and plasminogen activator inhibitor. The cDNA was subcloned in the pET3d expression vector, equipped with a histidine a ffinity tag at the N-terminus and expressed in Escherichia coli BL(21) DE3 pLysS. Recombinant WSZ1 from the soluble fraction was partially p urified on Ni-NTA agarose and MonoQ columns and shown to form SDS-stab le complexes with sc-chymotrypsin. Southern blots and amino acid seque ncing indicated that only few serpins are encoded by wheat, but at lea st three distinct genes are expressed in the grain. Cleavage experimen ts on a chymotrypsin column suggested a Gln-Gln reactive site bond not previously observed in inhibitory serpins.