Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Evaluation of a New Episomal Vector Based on the GAP Promoter for Structural Genomics in Pichia pastoris

  • Hong In-Pyo (Department of Bioengineering and Technology, Kangwon National University) ;
  • Anderson Stephen (Northeast Structural Genomics Consortium (NESG)) ;
  • Choi Shin-Geon (Department of Bioengineering and Technology, Kangwon National University)
  • Published : 2006.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

A new constitutive episomal expression vector, pGAPZ-E, was constructed and used for initial screening of eukaryotic target gene expression in Pichia pastoris. Two reporter genes such as beta-galactosidase gene and GFPuv gene were overexpressed in P. pastoris. The expression level of the episomal pGAPZ-E strain was higher than that of the integrated form when the beta-galactosidase gene was used as the reporter gene in P. pastoris X33. The avoiding of both the integration procedure and an induction step simplified the overall screening process for eukaryotic target gene expression in P. pastoris. Nine human protein targets from the Core 50, family of Northeast Structural Genomics Consortium (http://www.nesg.org), which were intractable when expressed in E. coli, were subjected to rapid screening for soluble expression in P. pastoris. HR547, HR919, and HR1697 human proteins, which had previously been found to express poorly or to be insoluble in E. coli, expressed in soluble form in P. pastoris. Therefore, the new episomal GAP promoter vector provides a convenient and alternative system for high-throughput screening of eukaryotic protein expression in P. pastoris.

Keywords

References

  1. Acton, T., K. Gunsalus, R. Xiao, L. Ma, J. Aramini, M. Baran, Y. Chiang, T. Climent, B. Cooper, N. Denissova, S. Douglas, J. Everett, C. Ho, D. Macapagal, P. Rajan, R. Shastry, L. Shih, G. Swapna, M. Wilson, M. Wu, M. Gerstein, M. Inouye, J. Hunt, and G. Montelione. 2005. Robotic cloning and protein production platform of the Northeast Structural Genomics Consortium. Methods Enzymol. 394:210-243 https://doi.org/10.1016/S0076-6879(05)94008-1
  2. Baneyx, F. 1999. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10: 411-421 https://doi.org/10.1016/S0958-1669(99)00003-8
  3. Boettner, M., B. Prinz, C. Holz, U. Stahl, and C. Lang. 2002. High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. J. Biotechnol. 99: 51-62 https://doi.org/10.1016/S0168-1656(02)00157-8
  4. Bottner, M. and C. Lang. 2004. High-throughput expression in microplate format in Pichia pastoris. Methods Mol. Biol. 267: 277-286
  5. Cha, H. J., N. G. Dalai, and W. E. Bentley. 2004. In vivo monitoring of intracellular expression of human interleukin-2 using green fluorescent protein fusion partner in Pichia pastoris. Biotechnol. Lett. 26: 1157-1162 https://doi.org/10.1023/B:BILE.0000035489.09417.d4
  6. Cereghino, J. and J. Cregg. 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24: 45-66 https://doi.org/10.1111/j.1574-6976.2000.tb00532.x
  7. Cregg, J., K. Barringer, A. Hessler, and K. Madden. 1985. Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 5: 3376-3385 https://doi.org/10.1128/MCB.5.12.3376
  8. Cregg, J., T. Vedvick, and W. Raschke. 1993. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (NY) 11: 905-910 https://doi.org/10.1038/nbt0893-905
  9. de Almeida, J. R., L. M. de Moraes, and F. A. Torres. 2005. Molecular characterization of the 3-phosphogly cerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris.Yeast 22: 725-737 https://doi.org/10.1002/yea.1243
  10. Faber, K., W. Harder, G Ab, and M. Veenhuis. 1995. Review: Methylotrophic yeasts as factories for the production of foreign proteins. Yeast 11: 1331-1344 https://doi.org/10.1002/yea.320111402
  11. Hohenblum, H., B. Gasser, M. Maurer, N. Borth, and D. Mattanovich. 2004. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol. Bioeng. 85: 367-375 https://doi.org/10.1002/bit.10904
  12. Holz, C, O. Hesse, N. Bolotina, U. Stahl, and C. Lang. 2002. A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae. Protein Exp. Purif. 25: 372-378 https://doi.org/10.1016/S1046-5928(02)00029-3
  13. Holz, C. and C. Lang. 2004. High-throughput expression in microplate format in Saccharomyces cerevisiae. Methods Mol. Biol. 267: 267-276
  14. Hunt, I. 2005. From gene to protein: A review of new and enabling technologies for multi-parallel protein expression. Protein Exp. Purif. 40: 1 -22 https://doi.org/10.1016/j.pep.2004.10.018
  15. International Human Genome Sequencing Consortium. 2001. Initial sequencing and analysis of the human genome. Nature 409: 860-921 https://doi.org/10.1038/35057062
  16. Invitrogen Corporation. 1997. A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris. Invitrogen Corporation, San Diego, U.S.A
  17. Lee, C, T. Williams, D. Wong, and G. Robertson. 2005. An episomal vector for screening mutant gene libraries in Pichia pastoris. Plasmid 54: 80-85 https://doi.org/10.1016/j.plasmid.2004.12.001
  18. Lee, J.-L, S.-N. Yang, C.-S. Park, D. Jeoung, and H.-Y. Kim. 2004. Purification and glycosylation pattern of human L-ferritin in Pichia pastoris. J. Microbiol. Biotechnol. 14: 68-73
  19. Lee, J.-S., J. Y, H.-J. Shin, Y-S. Kim, J.-K. Ahn, C.-K. Lee, H. Poo, and C.-J. Kim. 2005. Expression of hepatitis C virus structural proteins in Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 15:767-771
  20. Lueking, A., C. Holz, C. Gotthold, H. Lehrach, and D. Cahill. 2000. A system for dual protein expression in Pichia pastoris. Protein Exp. Purif. 20: 373-378
  21. Lueking, A., S. Horn, H. Lehrach, and D. Cahill. 2003. A dual expression vector allowing expression in E. coli and P. pastoris, including new modifications. Methods Mol. Biol. 205:31-42
  22. Macauley-Patrick, S., M. L. Fazenda, R. McNeil, and L. M. Harvey. 2005. Heterologous protein expression using the Pichia pastoris expression system. Yeast 22: 249-270 https://doi.org/10.1002/yea.1208
  23. Menendez, J., L. Hernandez, A. Banguela, and J. Pais. 2004. Functional production and secretion of the Gluconoacetobacter diazatrophicus fructose-releasing exo-Ievanase (LsdB) in Pichia pastoris. Enz. Microb. Technol. 34: 446-452 https://doi.org/10.1016/j.enzmictec.2003.11.018
  24. Mergulhao, Filipe J. M., G. A. Monteiro, J. M. S. Cabral, and M. A. Taipa. 2004. Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. J. Microbiol. Biotechnol. 14: 1-14
  25. Miller, J. H. 1972. Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  26. Prinz, B., J. Schultchen, R. Rydzewski, C. Holz, M. Boettner, U. Stahl, and C. Lang. 2004. Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics. J. Struct. Fund. Genomics 5: 29-44 https://doi.org/10.1023/B:JSFG.0000029207.13959.90
  27. Ro, H.-S., M.-S. Lee, M.-S. Hahm, H.-S. Bae, and B. H. Chung. 2005. Production of active carboxypeptidase Y of Saccharomyces cerevisiae secreted from methylotrophic yeast Pichia pastoris. J. Microbiol. Biotechnol. 15: 202-205
  28. Sunga, A. J. and J. M. Cregg. 2004. The Pichia pastoris formaldehyde dehydrogenase gene (PNOl) as a marker for selection of multicopy expression strains of P. pastoris. Gene 330: 39-47 https://doi.org/10.1016/j.gene.2003.12.015
  29. Tschopp, J., P. Brust, J. Cregg, C. Stillman, and T. Gingeras. 1987. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res. 15: 3859-3876 https://doi.org/10.1093/nar/15.9.3859
  30. Waterham, H., M. Digan, P. Koutz, S. Lair, and J. Cregg. 1997. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186: 37-44 https://doi.org/10.1016/S0378-1119(96)00675-0
  31. Yokoyama, S. 2003. Protein expression systems for structural genomics and proteomics. Curr. Opin. Chem. Biol. 7: 39-43 https://doi.org/10.1016/S1367-5931(02)00019-4
  32. Zupan, A. L., V. Trobec, V. Gaberc-Porekar, and V. Menart. 2004. High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles. J. Biotechnol. 109: 115-122 https://doi.org/10.1016/j.jbiotec.2003.11.013