Journal of Industrial Microbiology

, Volume 7, Issue 3, pp 197–201 | Cite as

High-level secretion of biologically active aprotinin from the yeastPichia pastoris

  • Thomas Vedvick
  • Richard G. Buckholz
  • Michael Engel
  • Mary Urcan
  • Jennifer Kinney
  • Sally Provow
  • Robert S. Siegel
  • Gregory P. Thill
Original Papers

Summary

A synthetic gene encoding aprotinin (bovine pancreatic trypsin, inhibitor) was fused to theSaccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site.Pichia pastoris strains were developed to'express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion.P. pastoris containing a single copy of the vector secreed approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipoten with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.

Key words

Secretion Proteolytic processing Protease inhibitor Pichia Yeast expression Aprotinin 

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Copyright information

© Society for Industrial Microbiology 1991

Authors and Affiliations

  • Thomas Vedvick
    • 1
  • Richard G. Buckholz
    • 1
  • Michael Engel
    • 1
  • Mary Urcan
    • 1
  • Jennifer Kinney
    • 1
  • Sally Provow
    • 1
  • Robert S. Siegel
    • 1
  • Gregory P. Thill
    • 1
  1. 1.The Salk Institute Biotechnology/Industrial Associates (SIBIA)San DiegoUSA

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