Sibia Neurosciences, Inc.

The Salk Institute Biotechnology Industrial Associates, Inc.

The Salk Institute

The Salk Institute Biotechnology/industrial Assoc, Inc.

Bradley D Howard

Martin Anthony Gleeson

Russell A Brierley

Gregory C Holtz

Geneva R Davis

The isolation and characterization of genes involved in proteolytic processing in species of the genus Pichia is described. The availability of such genes has enabled the generation of strains of Pichia which are deficient in proteolytic activity, which strains are useful as hosts for the expression of proteolytically sensitive recombinant products. The isolation and characterization of additional genes from species of the genus Pichia is also described, as well as uses therefore.

Genes which influence pichia proteolytic activity, and uses therefor

Insulin-like growth factor-1 (IGF-1), a naturally occurring, relatively short, single chain polypeptide, is prepared by growing methylotrophic yeast transformants containing in their genome at least one copy of DNA encoding IGF-1, in operational linkage with DNA encoding a signal sequence, which is effective for directing secretion of proteins from the host cells and which also includes the proteolytic processing site lys-arg and may include one or more glu-ala sequences. In preferred embodiments the signal sequence is the S. cerevisiae alpha mating factor pre-pro sequence. Expression of both the DNA encoding IGF-1 and the pre-pro signal sequence is regulated by a promoter region derived from a methanol responsive gene of a methylotrophic yeast. DNA constructs and recombinant methylotrophic yeast strains used for the expression and secretion of IGF-1 are also provided. For preferred embodiments, protease deficient Pichia pastoris strains are provided.

Production of insulin-like growth factor-1 in methylotrophic yeast cells

Genes which influence Pichia proteolytic activity, and uses therefor

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San Diego, CA, United States of America

Bradley D Howard