Cloning and expression of the gene encoding a novel proteinase from Tritirachium album limber

We have isolated the cDNA and the genomic clones encoding a novel serine proteinase, named proteinase T, from the fungus Tritirachium album Limber. The coding region of the gene is interrupted by two introns. The amino acid sequence of proteinase T as deduced from the nucleotide sequence is about 56% identical to that of proteinase K. Four cysteines are present in the mature proteinase, probably in the form of disulfide bonds. We have also purified the native proteinase from Tritirachium album Limber grown in the presence of 2% skim milk. Proteinase T is extremely stable at 50 degrees C. The thermal stability is not affected in the presence of 1% SDS either at pH 8.0 or 10.0. We have expressed the cDNA of proteinase T in Escherichia coli. The authenticity of the proteinase has been characterized by Western blotting and amino terminal analysis of the recombinant product. High level expression of proteinase T in E. coli as well as the refolding process to generate active proteinase will be discussed in detail.