Pichia pastoris, a popular protein expression system, is limited in its ability to secrete heterologous proteins. The PMR1 gene, the disruption of which is known to improve the secretion of prochymosin, human prourokinase, and human tissue plasminogen activator in Saccharomyces cerevisiae, was cloned from P. pastoris. The pmr 1 mutant in S. cerevisiae also displayed a slow growth phenotype when grown on low Ca2+ medium. The putative P. pastoris PMR1 gene, encoding for a 924 amino acid P-type Ca2+ ATPase, was disrupted in P. pastoris and the secretion of horseradish peroxidase (HRP) and β-galactosidase (β-gal) analyzed. Secreted HRP activity was determined using 3,3',5,5' tetramethylbenzidine (TMB) colorimetric assay and western analysis. β-gal expression and secretion was determined by western analysis. Secretion in P. pastorius Δpmr1 for both heterologous proteins showed no appreciable difference compared to wild type, nor did P. pastoris Δpmr1 display the slow growth phenotype seen in S. cerevisiae Δpmr1 (Rudolph H. et al., 1989).
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-1612 |
| Date | 01 January 2005 |
| Creators | Grove, Heather Lee |
| 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 |
Page generated in 0.2558 seconds