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.

Structure, secretion, and proteolysis study of MBP-containing heterologous proteins in Pichia pastoris

Author

Zhiguo Li, University of the Pacific

Date of Award

2010

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Andrew Franz

First Committee Member

Geoffrey Lin-Cereghino

Second Committee Member

Patrick Jones

Third Committee Member

Jianhua Ren

Fourth Committee Member

Xue Liang

Abstract

The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N -terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris , a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N -terminal partner to several C -terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C -terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C -terminal of the MBP domain, including the spacer and cargo regions, but the MEP domain could still act to enhance the secretion of certain cargo proteins. The attempt to identify the unknown protease was unsuccessful. However, in contrast to other fusion partners, MBP was secreted with the cargo when it was fused as a C -terminal peptide to an N -terminal cargo protein. These studies provide insights into the role of proteases and fusion partners in the secretory mechanism of P. pastoris , suggesting new strategies to optimize this expression system.

Pages

141

ISBN

9781124167428

Recommended Citation

Li, Zhiguo. (2010). Structure, secretion, and proteolysis study of MBP-containing heterologous proteins in Pichia pastoris. University of the Pacific, Dissertation - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/2415