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Title:Optimization of secretion of bovine pancreatic trypsin inhibitor from yeast
Author(s):Parekh, Rajesh N.
Doctoral Committee Chair(s):Wittrup, K. Dane
Department / Program:Chemical and Biomolecular Engineering
Discipline:Chemical and Biomolecular Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Engineering, Chemical
Abstract:As a single-celled microbial eucaryotic host for protein expression, the yeast Saccharomyces cerevisiae offers some of the advantages of both bacterial systems (high cell density fermentation technology) and eucaryotic systems (secretion of correctly folded and assembled proteins). One disadvantage however, is that secretion of foreign proteins is generally inefficient in yeast.
The eucaryotic secretory pathway provides a direct and efficient means of producing correctly folded and assembled proteins since the endoplasmic reticulum's proofreading apparatus ensures that misfolded proteins are not released to the medium. However, it has in general been difficult to attain the high levels of productivity characteristic of bacterial hosts (i.e., grams per liter of protein product) using mammalian and yeast secretion systems. A deeper understanding of the folding environment in the ER will enable rational engineering of efficient eucaryotic expression systems for pharmaceutical proteins. We have chosen bovine pancreatic trypsin inhibitor (BPTI), expressed and secreted in Saccharomyces cerevisiae, as a model system for such studies. BPTI was chosen as a model protein since it represents the class of proteins (small, non glycosylated and disulfide bonded) that are usually produced in yeast commercially. It is also useful as a model protein since its structure and folding kinetics have been exhaustively characterized in vitro.
Our studies have lead us to the following observations: (1) An optimal rate of synthesis exists for BPTI secretion and as synthesis increases beyond this level, secretion decreases to a level significantly below the maximum, concomitant with accumulation of misfolded BPTI in the ER and gross morphological changes in the cell. (2) Secretion efficiency of mutant forms of BPTI with Cys/Ala replacements correlates with thermodynamic stability, but not with in vitro folding or unfolding kinetics. (3) Perturbations in the ER environment can affect the yield of secreted product, though the effect is highly dependent on the aeration history of the culture.
Issue Date:1996
Rights Information:Copyright 1996 Parekh, Rajesh N.
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9712396
OCLC Identifier:(UMI)AAI9712396

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