The methylotrophic yeast, Pichia pastoris, has been used to successfully produce and express over 700 heterologous proteins. Currently, the only two dominant selectable markers available are the Zeocin and blasticidin resistance genes. This study focused on characterizing and developing the modified G418 selectable marker so it could be used for primary selection of single copy and multicopy strains. To demonstrate its use as a selectable marker the SLPI reporter gene was inserted into the Zeocin resistance vector, pPICZB, and the new G418 resistance vector, pKANa B. The resulting constructs were then transformed into P. pastoris yJC100 cells. Piggyback strains containing both resistance vectors were also constructed. Real-time PCR, spot western blot, western immunoblot, and ELISA analysis confirmed that the modified G418 resistant vector was comparable to the Zeocin resistant vector and that its capable of generating multicopy transformants. In addition, analysis of our piggyback strains showed an increase in SIPI production and in plasmid copy number when compared to their respective "mother
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-1735 |
| Date | 01 January 2009 |
| Creators | Moy, Allison |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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