Genes which influence pichia proteolytic activity, and uses therefor

A method for the expression of proteolytically sensitive recombinant product(s), comprising:

• contacting a Pichia host strain with a DNA fragment under conditions suitable for effecting site-directed integration of the DNA fragment into the genome of the host to produce a host strain deficient in proteolytic activity, wherein:
• the DNA fragment contains a modified form of a gene encoding a protein which, directly or indirectly, influences the proteolytic activity of the strain;
• the modification renders the gene incapable of producing functional product, or alters the ability of the gene product to influence the proteolytic activity of the strain;
• the gene that encodes the product that influences the proteolytic activity is selected from the group consisting of the Pichia PEP4 gene and the Pichia PRB-1 gene;
• the host strain is defective in at least one auxotrophic marker gene;
• and
• the host strain is transformed with at least one DNA fragment, comprising, in the reading frame direction of transcription, the following sequences of nucleotides:
• (i) a promoter region of a methanol-responsive gene of a methylotrophic yeast,
• (ii) a sequence of nucleotides encoding the proteolytically sensitive protein product;
• wherein said sequences of nucleotides are operationally associated with one another for transcription of the sequences of nucleotides that encode the product.