A recombinant wheat serpin with inhibitory activity

Søren K Rasmussen, Søren Weis Dahl, Anette Nørgård, Jørn Hejgaard

    Research output: Contribution to journalJournal articleResearchpeer-review

    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 alpha(1)-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 sc-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.
    Original languageEnglish
    JournalPlant Molecular Biology
    Volume30
    Issue number3
    Pages (from-to)673-677
    ISSN0167-4412
    DOIs
    Publication statusPublished - Feb 1996

    Keywords

    • cDNA sequence
    • chymotrypsin inhibitor
    • histidine-tag
    • protein Z
    • serpin
    • Triticum aestivum

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