Cloning and characterization of the Pichia Pastoris PMR1 gene

<p>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 &#946;-galactosidase (&#946;-gal) analyzed. Secreted HRP activity was determined using 3,3',5,5' tetramethylbenzidine (TMB) colorimetric assay and western analysis. &#946;-gal expression and secretion was determined by western analysis. Secretion in P. pastorius &Delta;pmr1 for both heterologous proteins showed no appreciable difference compared to wild type, nor did P. pastoris &Delta;pmr1 display the slow growth phenotype seen in S. cerevisiae &Delta;pmr1 (Rudolph H. et al., 1989).

Name thesis:degree_name

Master of Science (M.S.)

Level thesis:degree_level

Thesis - Pacific Access Restricted

Discipline thesis:degree_discipline

Biological Sciences

Year dc:date.available

2005

Author dc:creator

• Grove, Heather Lee

Contributors dc:contributor

• Geoff Lin-Cereghino

• Pichia pastoris
• Pichia Genetics
• Yeast fungi Biotechnology
• Molecular cloning
• Biology
• Life Sciences

Licence

• http://rightsstatements.org/vocab/InC/1.0/

Repository record dc:identifier

https://scholarlycommons.pacific.edu/uop_etds/613

OAI identifier oai:identifier

oai:scholarlycommons.pacific.edu:uop_etds-1612