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Biotechnology Letters

, Volume 27, Issue 5, pp 339–345 | Cite as

Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris

  • Manuel Mansur
  • Cecilia Cabello
  • Lester Hernández
  • José País
  • Laura Varas
  • Jorge Valdés
  • Yanet Terrero
  • Abdel Hidalgo
  • Liuba Plana
  • Vladimir Besada
  • Liudys García
  • Emilio Lamazares
  • Lila Castellanos
  • Eduardo Martínez
Article

Abstract

We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l1 when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.

Keywords

insulin multiple gene copies Pichia pastoris protein expression 

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Copyright information

© Springer 2005

Authors and Affiliations

  • Manuel Mansur
    • 1
  • Cecilia Cabello
    • 1
  • Lester Hernández
    • 1
  • José País
    • 1
  • Laura Varas
    • 1
  • Jorge Valdés
    • 1
  • Yanet Terrero
    • 1
  • Abdel Hidalgo
    • 1
  • Liuba Plana
    • 2
  • Vladimir Besada
    • 1
  • Liudys García
    • 1
  • Emilio Lamazares
    • 1
  • Lila Castellanos
    • 1
  • Eduardo Martínez
    • 1
  1. 1.Center for Genetic Engineering and Biotechnology (CIGB)HavanaCuba
  2. 2.National Institute of Fundamental Research in Tropical AgricultureSantiago de las VegasCuba

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