Abstract
A full-length clone encoding the wheat (Triticum aestivum L.) serpin WSZ1 was isolated from a cDNA library based on mRNA from immature grain. The 398 amino acid sequence deduced from the cDNA was corroborated by sequencing CNBr peptides of WSZ1 purified from resting grain. WSZ1 belongs to the subfamily of protein Z-type serpins and the amino acid sequence is 70% identical with the barley serpins BSZ4 and BSZx and 27–33% identical with human serpins such as α1-proteinase inhibitor, antithrombin III, and plasminogen activator inhibitor. The cDNA was subcloned in the pET3d expression vector, equipped with a histidine affinity tag at the N-terminus and expressed in Escherichia coli BL(21) DE3 pLysS. Recombinant WSZ1 from the soluble fraction was partially purified on Ni-NTA agarose and MonoQ columns and shown to form SDS-stable complexes with α-chymotrypsin. Southern blots and amino acid sequencing indicated that only few serpins are encoded by wheat, but at least three distinct genes are expressed in the grain. Cleavage experiments on a chymotrypsin column suggested a Gln-Gln reactive site bond not previously observed in inhibitory serpins.
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References
Brandt A, Svendsen I, Hejgaard J: A plant serpin gene. Structure, organization and expression of the gene encoding barley protein Z4. Eur J Biochem: 499–505 (1990).
Cooperman BS, Stavridi E, Nickbarg E, Rescorla E, Schechter NM, Rubin H. Antichymotrypsin interaction with chymotrypsin. J Biol Chem 268: 23616–23625 (1993).
Hejgaard J: Purification and properties of protein Z, a major albumin of barley endosperm. Physiol Plant 54: 174–182 (1982).
Hejgaard J, Rasmussen SK, Brandt A, Svendsen I: Sequence homology between barley endosperm protein Z and protease inhibitors of the α1-antitrypsin family. FEBS Lett 180: 89–94 (1985).
Hook VYH, Purviance RT, Azaryan AV, Hubbard G, Krieger TJ: Purification and characterization of α1-antichymotrypsin-like protease inhibitor that regulates prohormone thiol protease involved in enkephalin precursor processing. J Biol Chem 268: 20570–20577 (1993).
Hopkins PCR, Carrell RW, Stone SR: Effects of mutations in the hinge region of serpins. Biochemistry 32: 7650–7657 (1993).
Lundgard R, Svensson B: A 39 kD barley seed protein of the serpin superfamily inhibits α1-chymotrypsin. Carlsberg Res Commun 54: 173–180 (1989).
Patston PA, Gettins PGW: A database of recombinant wild-type and mutant serpins. Tromb Haemost 72: 166–179 (1994).
Rasmussen SK: A gene coding for a new plant serpin. Biochim Biophys Acta 1172: 151–154 (1993).
Rosekrands I, Hejgaard J, Rasmussen SK, Bjørn SE: Serpins from wheat grain. FEBS Lett 343: 75–80 (1994).
Schulze AJ, Huber R, Bode W, Engh RA: Structural aspects of serpin inhibition. FEBS Lett 344: 117–124 (1994).
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of T7 RNA polymerase to direct expression. Meth Enzymol 185: 60–89 (1990).
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Rasmussen, S.K., Dahl, S.W., Nørgård, A. et al. A recombinant wheat serpin with inhibitory activity. Plant Mol Biol 30, 673–677 (1996). https://doi.org/10.1007/BF00049343
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DOI: https://doi.org/10.1007/BF00049343