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

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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 l−1 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.

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References

  • DM Becker L Guarente (1991) ArticleTitleHigh-efficiency transformation of yeast by electroporation. Methods Enzymol 194 182–187 Occurrence Handle10.1016/0076-6879(91)94015-5 Occurrence Handle1:CAS:528:DyaK3MXit1akur0%3D

    Article  CAS  Google Scholar 

  • RA Brierley (1998) ArticleTitleSecretion of recombinant human insulin-like growth factor I (IGF-I) Methods Mol. Biol. 103 149–177 Occurrence Handle1:CAS:528:DyaK1cXjslCltrg%3D

    CAS  Google Scholar 

  • RA Brierley GR Davis GC Holtz MA Gleeson BD Howard (1997) ArticleTitleProduction of insulin-like growth factor-1 in methylotrophic yeast cells US Patent 5 612–198

    Google Scholar 

  • LR Castellanos-Serra C Fernández E Hardy V Huerta (1996) ArticleTitleA procedure for protein elution from reverse-stained polyacrylamide gels applicable at the low picomole level: an alternative route to the preparation of low abundance proteins for microanalysis Electrophoresis 17 1564–1572 Occurrence Handle1:CAS:528:DyaK28XmslCksrk%3D

    CAS  Google Scholar 

  • GP Cereghino JM Cregg (1999) ArticleTitleApplication of yeast in biotechnology: protein production and genetic analysis Curr. Opin. Biotechnol. 10 422–427 Occurrence Handle1:STN:280:DyaK1MvjvFGltQ%3D%3D

    CAS  Google Scholar 

  • JJ Clare FB Rayment SP Ballantine K Sreekrishna MA Romanos (1991) ArticleTitleHigh-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene Biotechnology 9 455–460 Occurrence Handle1:CAS:528:DyaK38XnsleqsQ%3D%3D

    CAS  Google Scholar 

  • JJ Clare MA Romanos F Rayment JE Rowedder MA Smith MM Payne K Sreekrishna CA. Henwood (1991) ArticleTitleProduction of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastorisstrains containing multiple gene copies Gene 105 205–212 Occurrence Handle1:CAS:528:DyaK3MXmslamt78%3D

    CAS  Google Scholar 

  • H Cox D Mead P Sudbery RM Eland I Mannazzu L Evans (2000) ArticleTitleConstitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter Yeast 16 1191–1203 Occurrence Handle1:CAS:528:DC%2BD3cXnt1ymtLo%3D

    CAS  Google Scholar 

  • JM Cregg JL Cereghino J Shi DR Higgins (2000) ArticleTitleRecombinant protein expression in Pichia pastoris Mol. Biotechnol. 16 23–52 Occurrence Handle1:CAS:528:DC%2BD3cXot1agtLk%3D

    CAS  Google Scholar 

  • Feng YM, Zhang BY, Zhang YS, Hiroshi F (1997). Secretory expression of porcine insulin precursor in Kluyveromyces lactis and its conversion into human insulin. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 29: 129–134

  • G Gellissen K Melber (1996) ArticleTitleMethylotrophic yeast Hansenula polymorpha as production organism for recombinant pharmaceuticals. Arzneimittelforschung 46 943–948 Occurrence Handle1:CAS:528:DyaK28XlvFelsr0%3D

    CAS  Google Scholar 

  • T Kjeldsen A Frost-Petterson M Hach (1999) ArticleTitleSecretory expression and characterization of insulin in Pichia pastoris Biotechnol. Appl. Biochem. 29 79–86 Occurrence Handle1:CAS:528:DyaK1MXhsFyqtbg%3D

    CAS  Google Scholar 

  • S Linder M Schliwa E Kube-Granderath (1996) ArticleTitleDirect PCR screening of Pichia pastoris clones Biotechniques 20 980–982 Occurrence Handle1:CAS:528:DyaK28XjtlGmsbg%3D

    CAS  Google Scholar 

  • AF Mayer K Hellmuth H Schlieker R Lopez-Ulibarri S Oertel U Dahlems AW Strasser AP Loon Particlevan (1999) ArticleTitleAn expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha Biotechnol. Bioeng. 63 373–381 Occurrence Handle1:CAS:528:DyaK1MXhvFymtLo%3D

    CAS  Google Scholar 

  • JM País L Varas J Valdés C Cabello L Rodríguez M Mansur (2003) ArticleTitleModeling of mini-proinsulin production in Pichia pastoris using the AOX promoter Biotechnol. Lett. 25 251–255

    Google Scholar 

  • H Schägger G Jagow Particlevon (1987) ArticleTitleTricine-sodium dodecyl sulfate – polyacrilamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal. Biochem. 166 368–379

    Google Scholar 

  • A Vassileva DA Chugh S Swaminathan N Khanna (2001) ArticleTitleExpression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter J. Biotechnol. 88 21–35 Occurrence Handle1:CAS:528:DC%2BD3MXjslSls78%3D

    CAS  Google Scholar 

  • T Vedvick RG Buckholz M Engel M Urcan J Kinney S Provow RS Siegel GP Thill (1991) ArticleTitleHigh-level secretion of biologically active aprotinin from the yeast Pichia pastoris J. Ind. Microbiol. 7 197–201 Occurrence Handle1:CAS:528:DyaK3MXltlCjtL4%3D

    CAS  Google Scholar 

  • Y Wang ZH Liang YS Zhang SY Yao YG Xu YH Tang SQ Zhu DF Cui YM Feng (2001) ArticleTitleHuman insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product Biotechnol. Bioeng. 73 74–79 Occurrence Handle1:CAS:528:DC%2BD3MXitFGqu7w%3D

    CAS  Google Scholar 

  • MWT Werten TJ Bosch Particlevan den RD Wind H Mooibroek FA Wolf Particlede (1999) ArticleTitleHigh-yield secretion of recombinant gelatins by Pichia pastoris Yeast 15 1087–1096 Occurrence Handle1:CAS:528:DyaK1MXlsVyktro%3D

    CAS  Google Scholar 

  • S Wu RD Fallon MS Payne (1999) ArticleTitleEngineering Pichia pastoris for stereoselective nitrile hydrolysis by co-producing three heterologous proteins Appl. Microbiol. Biotechnol. 52 186–190 Occurrence Handle1:CAS:528:DyaK1MXmtFKhtr4%3D

    CAS  Google Scholar 

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Authors and Affiliations

  1. Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, Havana, 11600, Cuba

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

  2. National Institute of Fundamental Research in Tropical Agriculture, Santiago de las Vegas, Havana, 17200, Cuba

    Liuba Plana

Authors
  1. Manuel Mansur
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  2. Cecilia Cabello
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  3. Lester Hernández
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  4. José País
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  5. Laura Varas
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  6. Jorge Valdés
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  7. Yanet Terrero
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  8. Abdel Hidalgo
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  9. Liuba Plana
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  10. Vladimir Besada
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  11. Liudys García
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  12. Emilio Lamazares
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  13. Lila Castellanos
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  14. Eduardo Martínez
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Correspondence to Manuel Mansur.

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Mansur, M., Cabello, C., Hernández, L. et al. Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris . Biotechnol Lett 27, 339–345 (2005). https://doi.org/10.1007/s10529-005-1007-7

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  • DOI: https://doi.org/10.1007/s10529-005-1007-7

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