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Expression, extraction and activity testing of the metacaspase Scp3
Author(s) -
Gagliardi Connor,
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.1034.2
Subject(s) - biochemistry , chemistry , enzyme , yeast , escherichia coli , gene
Apoptosis, a fundamental characteristic of multicellular eukaryotic organisms, is a key component in maintaining organismal integrity. Proteins called metacaspases regulate apoptosis in plants, protozoans and fungi. These proteases are responsible for cleavage of other proteins during programmed cell death. Metacaspases preferentially cleave after basic residues in the P1 subsite. The substrate specificity at the P2 and P3 subsites varies for the metacaspases in plants and fungi. But, metacaspase specificity been studied in only a few plants and in two fungal organisms ( S. cerevisiae and S. pombe) . Our lab has been studying the five metacaspases from Schizophyllum commune , a split gill mushroom, to better understand the substrate specificity and function of these enzymes. The metacaspase Scp3 has been successfully cloned from the S. commune genome, and transformed into and expressed from Escherichia coli . Using two rounds of nickel affinity chromatography we have obtained protein that is >;95% pure. Current activity assays have shown significant enzymatic activity of Scp3 with the substrate FITC‐Casein compared to a crude cell extract without Scp3. Our lab is currently working to optimize the expression of Scp3 via autoinduction in various media and at different growth temperatures. Additionally, optimization of the extraction and assay conditions for the metacaspase are being completed in conjunction with one another. Optimization of extraction will provide us with more stable and active protein that can be tested for enzymatic activity. Once optimization of all processes is completed for Scp3 the knowledge can be transferred to the other four metacaspases of S. commune.