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Genotyping of recombinant Pichia pastoris strains

Cellular & Molecular Biology Letters volume 11, pages 348–359 (2006)Cite this article

Abstract

A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns. A clonal variation was obtained after the transformation process due to genetic differences generated during the transformation event of the host strain. Furthermore, the cluster analysis showed that the transformants with lesser genetic differences with respect to the P. pastoris host strain are the recombinant strains with the highest level of recombinant protein production.

Abbreviations

AFLP:

amplified-fragment length polymorphism

AOX1:

alcohol oxidase 1 gene

BMG:

growth medium: 100 mM potassium phosphate, pH 6.0, 0.4 mg/l biotin, 1.34% YNB, 1% glycerol

BMM:

expression medium: 100 mM potassium phosphate, pH 6.0, 0.4 mg/l biotin, 1.34% YNB, 0.75% (v/v) methanol

DMT-off:

without dimethoxytrityl

n.d.:

not determined

PCR:

polymerase chain reaction

RDB:

1 M sorbitol, 1.34% YNB, 2% dextrose, 0.4 mg/l biotin, 0.005% amino acids without histidine

r2 :

coefficient of determination

RP:

recombinant protein

SDS:

sodium dodecyl sulphate

TE buffer:

10 mM Tris-Cl, 1 mM EDTA, pH 8.0

TP:

total protein

XA-1 and XA-2:

XhoI site cohesive-ended complementary oligonucleotides to generate the XhoI restriction fragment adapter

XP-G:

PCR primer used in the AFLP reactions directed at the adapter tail and with a G as selective nucleotide at the 3′-end

YNB:

yeast nitrogen base

YPD:

1% yeast extract, 2% peptone, 2% dextrose

References

  1. Romanos, M.A. Advances in the use of Pichia pastoris for high level gene expression. Curr. Opin. Biotechnol. 6 (1995) 527–533.

    Article  CAS  Google Scholar 

  2. Escamilla-Treviño, L.L., Viader-Salvadó, J.M. and Guerrero-Olazarán M. Producción de proteínas recombinantes en Pichia pastoris. CIENCIA UANL 2 (1999) 27–33.

    Google Scholar 

  3. Cregg, J.M., Barringer, K.J., Hessler, A.Y. and Madden, K.R. Pichia pastoris as a host for transformations. Mol. Cell. Biol. 5 (1985) 3376–3385.

    PubMed  CAS  Google Scholar 

  4. Clare, J.J., Rayment, F.B., Ballantine, S.P., Sreekrishna, K. and Romanos M.A. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology 9 (1991) 455–60.

    Article  PubMed  CAS  Google Scholar 

  5. Clare, J.J., Sreekrishna, K. and Romanos M. Expression of tetanus toxin fragment C. Methods Mol. Biol. 103 (1998) 193–208.

    PubMed  CAS  Google Scholar 

  6. Brankamp, R.G., Sreekrishna, K., Smith, P.L., Blankenship, D.T. and Cardin, A.D. Expression of a synthetic gene encoding the anticoagulant-antimetastatic proteinghilanten by the methylotropic yeast Pichia pastoris. Protein Expr. Purif. 6 (1995) 813–820.

    Article  PubMed  CAS  Google Scholar 

  7. Romanos, M., Scorer, C., Sreekrishna, K. and Clare, J. The generation of multicopy recombinant strains. in: Pichia Protocols. Methods in Molecular Biology. (Higgins, D.R. and Cregg, J.M., Eds.) Vol. 103, Humana Press Inc, Totowa, NJ, 1998, 55–72.

    Chapter  Google Scholar 

  8. Sreekrishna, K., Brankamp, R.G., Kropp, K.E., Blankenship, D.L., Tsay, J.-T., Smith, P.L., Wierschke, J.D., Subramaniam, A. and Birkenberger, L.A. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190 (1997) 55–62.

    Article  PubMed  CAS  Google Scholar 

  9. Scorer, C.A., Buckholz, R.G., Clare, J.J. and Romanos, M.A. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene 136 (1993) 111–119.

    Article  PubMed  CAS  Google Scholar 

  10. Scorer, C.A., Clare J.J., Macombie, R., Romanos, M.A. and Sreekrishna, K. Rapid selection using G418 of high copy number transformats of Pichia pastoris for high-level foreign gene expression. Biotechnology 12 (1994) 181–184.

    Article  PubMed  CAS  Google Scholar 

  11. Vos, P., Hogers, R., Bleeker, M., Reijans, V., Van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23 (1995) 4407–4414.

    PubMed  CAS  Google Scholar 

  12. Lin, J.J. and Kuo, J. AFLP: a novel PCR-based assay for plant and bacterial DNA fingerprinting. Focus 17 (1995) 66–70.

    Google Scholar 

  13. Becker, J., Vos, P., Kuiper, M., Salamini, F. and Heun, V. Combined mapping of AFLP and RFLP markers in barley. Mol. Gen. Genet. 249 (1995) 65–73.

    Article  PubMed  CAS  Google Scholar 

  14. Janssen, P., Coopman, R., Huys, G., Swings, J., Bleeker, M., Vos, P., Zabeau, M. and Kersters, K. Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy. Microbiol. 142 (1996) 1881–1893.

    Article  CAS  Google Scholar 

  15. Ganter, P.F. and de Barros-Lopes, M. The use of anonymous DNA markers in assessing worldwide relatedness in the yeast species Pichia kluyveri Bedford and Kudrjavzev. Can. J. Microbiol. 46 (2000) 967–980.

    Article  PubMed  CAS  Google Scholar 

  16. Anonymous. Pichia expression kit: a manual of methods for expression of recombinant proteins in Pichia pastoris. Invitrogen. Available from: URL: http://www.invitrogen.com/content/sfs/manuals/pich_man.pdf

  17. Cregg, J.M. and Russell, K.A. Transformation. in: Pichia Protocols. Methods in Molecular Biology. (Higgins, D.R. and Cregg, J.M., Eds.) Vol. 103, Humana Press Inc, Totowa, NJ, 1998, 27–39.

    Chapter  Google Scholar 

  18. Escamilla-Treviño, L.L., Viader-Salvadó J.M., Barrera-Saldaña H.A. and Guerrero-Olazarán, M. Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris. Biotechnol. Lett. 22 (2000) 109–114.

    Article  Google Scholar 

  19. Hecker, K.H. and Roux, K.H. High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. BioTechniques 20 (1996) 478–485

    PubMed  CAS  Google Scholar 

  20. Felsenstein, J. PHYLIP Phylogeny Inference Package (Version 3.2). Cladistics 5 (1989) 164–166.

    Google Scholar 

  21. Sneath, P.H.A. and Sokal, R.R. Numerical Taxonomy: the principles and practice of numerical classification. W.H. Freeman and Company, San Francisco, 1973, 230–234.

    Google Scholar 

  22. Sreekrishna, K. Strategies for optimizing protein expression and secretion in the methylotrophic yeast Pichia pastoris. in: Industrial microorganisms: basic and applied molecular genetics. (Balz, R.H., Hegeman, G.D. and Skatrud, P.L., Eds.) American Society for Microbiology, Washington, D.C., 1993, 119–126.

    Google Scholar 

  23. Knorr, C., Cheng, H.H., and Dodgson, J.B. DNA cloning and sequence analysis of chicken AFLP. Anim. Genet. 32 (2001) 156–159.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Instituto de Biotecnología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León. Ave. Pedro de Alba s/n, Col. Ciudad Universitaria, 66450, San Nicolás de los Garza, N.L., Mexico

    José M. Viader-Salvadó, Eddy L. Cab-Barrera, Luis J. Galán-Wong & Martha Guerrero-Olazarán

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  1. José M. Viader-Salvadó
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  2. Eddy L. Cab-Barrera
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  3. Luis J. Galán-Wong
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  4. Martha Guerrero-Olazarán
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Correspondence to José M. Viader-Salvadó.

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Viader-Salvadó, J.M., Cab-Barrera, E.L., Galán-Wong, L.J. et al. Genotyping of recombinant Pichia pastoris strains. Cell Mol Biol Lett 11, 348–359 (2006). https://doiorg.publicaciones.saludcastillayleon.es/10.2478/s11658-006-0029-z

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Cellular & Molecular Biology Letters

ISSN: 1689-1392

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