Cloning & Studies of Neurospora Alkaline Protease

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We have demonstrated heterologous protein expression in Neurospora crassa using two promoters - the glucose repressible promoter of grg-1 and the constitutive promoter of the B-tubulin. Neurospora transformants expressing a bovine preprochymosin cDNA accumulate soluble, enzymatically active mature chymosin in the culture medium. One important element of heterologous protein expression systems is the appropriate host strain, for which protease deficient mutants are often superior. Neurospora secretes an alkaline protease in the presence of protein in the culture medium when also starved for carbon, nitrogen, or sulfur. When induced, the alkaline protease comprises 40% of secreted proteins. We are cloning cDNA and the genomic locus encoding the N. crassa secreted alkaline protease in order to develop an improved host and additional expression vectors for expression of heterologous proteins. %%% The development of microbial systems for expression of heterologous proteins has enabled the isolation of greater amounts of scarce proteins, often in greater purity than is possible from the original source. Proteins so expressed have applications ranging from industrial use, through protein biochemistry research, to human therapeutics. These clones will be used subsequently to create alkaline protease deficient disruption mutantions and to add the protease promoter and secretion signal peptide to our expression vector repertoire.