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Expression of a metacaspase from the fungus Schizophyllum commune
Author(s) -
Loew Ethan Bailey,
Fox Kristin M
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1034.1
Subject(s) - schizophyllum commune , escherichia coli , histidine , active site , chemistry , enzyme , biochemistry , protein subunit , caspase , biology , programmed cell death , gene , apoptosis
Metacaspases are a family of arginine/lysine specific peptidases known to induce programmed cell death in plants, protists, and fungi. Current research suggest that metacaspases contain a cysteine/histidine catalytic dyad in their active site, are calcium activated, and do not necessarily require proteolytic processing for catalytic activity. No specific substrate of metacaspases has been identified. To learn more about the structure and function of metacaspases, we are interested in expressing scp3, one of five putative metacaspases from the fungus Schizophyllum commune . Previous work in our lab has shown that the metacaspases are difficult to express in Escherichia coli . Because of similar difficulties with expression of active caspases, most are expressed as their individual p10 and p20 subunits, and then unfolded, combined, and refolded to produce active protein. To test this method with metacaspases, the p10 and p20 subunits of scp3 were amplified individually and cloned into pGEM® T‐Easy (Promega). The p10 and p20 subunits were then subcloned into the expression vector pQE‐80L (Qiagen) and successfully expressed in E. coli M15 cells. Work continues to fold the subunits and quantify their enzymatic activity.