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Optimization of expression of Schizophyllum commune metacaspase gene scp1 via codon‐optimization, prodomain removal, and autoinduction
Author(s) -
Major Ajay,
Fox Kristin M
Publication year - 2012
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.26.1_supplement.614.1
Subject(s) - proteases , caspase , chemistry , biochemistry , gene , codon usage bias , histidine , gene expression , homologous recombination , enzyme , microbiology and biotechnology , biology , programmed cell death , apoptosis , genome
In plants, fungi, and protists, programmed cell death is controlled by a family of proteases known as metacaspases, which appear to be homologous to aspartate‐specific mammalian caspases with high similarity of primary sequence and conservation of the cysteine‐histidine catalytic dyad. As proteases, metacaspases also contain N‐terminal prodomains that are cleaved off during proteolytic activation of the enzyme. However, metacaspases are arginine and lysine‐specific and thus have different self‐cleaving mechanisms and substrate interactions during programmed cell death. Expression and purification of S. commune metacaspase protein Scp1 is important for understanding these apoptotic mechanisms, but previous attempts at expression of the complete scp1 gene in E. coli were unsuccessful. Codon‐optimization of the scp1 gene resulted in no increase in expression. However, removal of the prodomain of codon‐optimized scp1 via PCR resulted in successful expression of Scp1 protein. Preliminary experiments with autoinduction, an expression technique in E. coli that utilizes glucose/lactose‐rich media, have shown increased expression of soluble Scp1 protein compared to IPTG‐induced overexpression.