No abstract

Metacaspases are Arg/Lys specific heterodimeric peptidases that are composed of a p10 and a p20 subunit that induce cell death cascades in fungi, plants and protozoa, making them novel drug‐targets. The fungus Schizophyllum commune contains five genes that code for metacaspases, of which only one, Scp3, has been expressed completely in Escherichia coli. Our overall aim is to produce soluble, active Scp3, however the Scp3 expressed in E. coli has been insoluble and inactive, compelling us to investigate a variety of different methods of expression. Homologous caspase proteins have been successfully produced via expression of the individual p10 and p20 subunits, that are then unfolded, combined and refolded into the active protein. Both subunits have already been cloned into the expression vector, pQE80/82(Qiagen), p20 expression was successful, and expression of p10 is underway in E. coli. In a parallel project we are attempting to express the full‐length Scp3 in E. coli and yeast. The full scp3 gene has been cloned into the pEQ80(Qiagen) and expressed in E. coli. After unfolding in guanidine HCl and refolding by dilution, small‐scale experiments have yielded soluble Scp3. Scp3 has also been cloned into the pYES vector (Invitrogen) and transformed into brewer's yeast, but no expression of Scp3 is observed when yeast are induced with galactose. Our aim is to produce soluble and active Scp3 that can be isolated for structural and functional analysis.