The Salk Institute Biotechnology Industrial Associates, Inc.

The Salk Institute

The Salk Institute Biotechnology/industrial Assoc, Inc.

Gregory C Holtz

Russell A Brierley

Geneva R Davis

Martin Anthony Gleeson

Bradley D Howard

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

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 a DNA sequence operably encoding IGF-1, in operational association with a DNA sequence encoding the S. cerevisiae alpha mating factor pre-pro sequence (including the proteolytic processing site: lys-arg), both under the regulation of a promoter region of a gene of a methylotrophic yeast, under conditions allowing expression of said DNA sequences, and secretion of IGF-1 into the culture medium. Also disclosed are novel DNA fragments and novel recombinant yeast strains which are useful in the practice of the present invention.

Method for the recovery and purification of intact, correctly-folded, monomeric insulin-like growth factor-1 peptide from large volumes of IGF-1-containing medium are described, comprising a series of adsorption-desorption steps employing a combination of cation exchange and hydrophobic interaction adsorbents. Product IGF-1 peptide is highly purified and suitable for use in a variety of clinical applications.

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San Diego, CA, United States of America

Gregory C Holtz