Applied Microbiology and Biotechnology

, Volume 48, Issue 4, pp 480–486 | Cite as

Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris

  • M. C. Loewen
  • X. Liu
  • P. L. Davies
  • A. J. Daugulis
ORIGINAL PAPER

Abstract

Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Muts clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut+ clones. The mixed-feeding/Muts strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l.

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Copyright information

© Springer-Verlag Berlin Heidelberg 1997

Authors and Affiliations

  • M. C. Loewen
    • 1
  • X. Liu
    • 2
  • P. L. Davies
    • 1
  • A. J. Daugulis
    • 2
  1. 1.Department of Biochemistry, Queen's University, Kingston, Ontario, Canada, K7L 3N6CA
  2. 2.Department of Chemical Engineering, Queens University, Kingston, Ontario, Canada, K7L 3N6 Tel.: (613)545 2784 Fax: (613)545 6637 e-mail: Daugulis@chee.queensu.caCA

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