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

Joan Lin-Cereghino

First Committee Member

Doug Risser

Second Committee Member

Der Thor

Abstract

Pichia pastoris is a methylotrophic yeast that has been used in both research and industrial settings for recombinant protein expression due to the ease of genetically modifying its genome, its ability to grow to large densities in inexpensive media, and its capability to perform posttranslational modifications. Multiple tools such as the cis -acting factors MATα secretion signal and MBP fusion partners, and trans-acting modifications such as the bgs mutants have increased heterologous protein secretion. Although these techniques have already been used, their effects on the protein secretory pathway have yet to be elucidated. In this study, fluorescence microscopy was used to compare the localization of proteins expressed with the mutated MATα with the deletion of amino acids 57-70 to the wild type MATα secretion signal. Additional fluorescence microscopy was completed to visualize the localization of MBP-EGFP and EGFP-MBP fusion proteins and their spatial relativity to organelle markers. EGFP-MBP was used to further distinguish the properties of multiple bgs mutants. Additionally, secreted lipase activity levels were evaluated in bgs13 strains expressing either the wild type or the mutated MAT&agr; signal peptide. The results indicated that regardless of their differences, the MATα secretion signals and bgs mutants transported their cargo proteins through similar pathways within the cells. The results of the MBP fusion proteins suggest that the arrangement of MBP significantly influences protein secretion and localization. Lastly, the bgs13 mutant with MATα secretion signals demonstrated that lipase activity increased additively when cis- and trans -acting mutations were combined. Ultimately, these results can provide better understanding of each modified factor and the protein sorting pathway, leading to potential techniques that optimize protein secretion in P. pastoris .

Pages

77

ISBN

9781321359893

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