Campus Access Only

All rights reserved. This publication is intended for use solely by faculty, students, and staff of University of the Pacific. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, now known or later developed, including but not limited to photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author or the publisher.

Date of Award

2014

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Der Thor

First Committee Member

Geoff Lin-Cereghino

Second Committee Member

Douglas Risser

Abstract

Pichia pastoris is a methylotrophic yeast that has been used widely in biological and industrial researches. P. pastoris is able to produce from milligram to gram quantities of protein. In this study, we examined the effects of calcium and magnesium on growth, heterologous protein expression, and calcium homeostasis in P. pastoris . The divalent cations calcium and magnesium are responsible for diverse roles in in the function of the cells in eukaryotes. Although it is known that calcium is responsible for many biological functions in eukaryotes, there is a limited understanding of the calcium homeostasis in P. pastoris . In this study, we found that addition of calcium and magnesium to yeast extract peptone dextrose (YPD) does not increase the cell growth of wild type P. pastoris . However, the original concentrations of calcium and magnesium in YPD were critical to cells, as removing calcium and magnesium using EGTA and EDTA, respectively, decreased the cell growth of wild type P. pastoris . Changes to cytoplasmic calcium concentration in P. pastoris was studied using the fluorescent calcium sensitive dye, indo-1 (10 μM) and fluorescent spectrophotometer. The intracellular calcium concentration increased with addition of calcium chloride, magnesium chloride, and phosphate buffered saline (PBS). PBS (standard 1x) induced a significantly higher increase in intracellular calcium concentration compared to inductions with calcium chloride (1.2 mM). Our results showed that the addition of calcium and magnesium into the growth medium did not increase the expression of alcohol oxidase-1 driven β-galactosidase expression. However, calcium and magnesium may still play crucial roles in protein expression as EDTA and EGTA caused an increase in β-galactosidase expression as indicated by higher β-galactosidase activity. Our findings suggest that EDTA and EGTA may also increase the expression of other heterologous proteins in P. pastoris .

Pages

65

ISBN

9781321359763

To access this thesis/dissertation you must have a valid pacific.edu email address and create an account for Scholarly Commons.

Find in ProQuest

Share

COinS

If you are the author and would like to grant permission to make your work openly accessible, please email