Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate‐limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate‐limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source. © 2004 Wiley Periodicals, Inc.

[1]  J. Sambrook,et al.  A 22 bp cis‐acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins. , 1992, The EMBO journal.

[2]  M. Meagher,et al.  High cell-density fermentation. , 1998, Methods in molecular biology.

[3]  U. Stahl,et al.  Intracellular transport of a heterologous membrane protein, the human transferrin receptor, in Saccharomyces cerevisiae , 2003, International microbiology : the official journal of the Spanish Society for Microbiology.

[4]  L. Thim,et al.  Secretion and processing of insulin precursors in yeast. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Peter Neubauer,et al.  Limiting factors in Escherichia coli fed-batch production of recombinant proteins. , 2003, Biotechnology and bioengineering.

[6]  T. Aizawa,et al.  Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system. , 2001, Protein engineering.

[7]  C. Masson,et al.  Optimization of the production of a honeybee odorant-binding protein by Pichia pastoris. , 1999, Protein expression and purification.

[8]  J. Sambrook,et al.  The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins , 1988, Nature.

[9]  V. Smirnov,et al.  Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum , 2002, BMC Molecular Biology.

[10]  K D Wittrup,et al.  Protein Folding Stability Can Determine the Efficiency of Escape from Endoplasmic Reticulum Quality Control* , 1998, The Journal of Biological Chemistry.

[11]  S. Swaminathan,et al.  Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. , 2001, Protein expression and purification.

[12]  K. A. Walsh,et al.  [3] Serine proteases , 1970 .

[13]  P. Walter,et al.  Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. , 2001, Current opinion in cell biology.

[14]  J J Clare,et al.  Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. , 1991, Gene.

[15]  Francis J Doyle,et al.  Decreased Protein Expression and Intermittent Recoveries in BiP Levels Result from Cellular Stress during Heterologous Protein Expression in Saccharomyces cerevisiae , 2002, Biotechnology progress (Print).

[16]  K. Kobayashi,et al.  High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation. , 2000, Journal of bioscience and bioengineering.

[17]  R. Armitage,et al.  Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants. , 1997, Gene.

[18]  Ronald T. Raines,et al.  Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments , 1998, Nature Biotechnology.

[19]  A. Dickson,et al.  Endoplasmic reticulum signaling as a determinant of recombinant protein expression , 2003, Biotechnology and bioengineering.

[20]  J. Cregg,et al.  Heterologous protein expression in the methylotrophic yeast Pichia pastoris. , 2000, FEMS microbiology reviews.

[21]  J. Cregg,et al.  Recombinant protein expression in Pichia pastoris , 2000, Molecular biotechnology.

[22]  J. Brodsky,et al.  Proteasome-dependent endoplasmic reticulum-associated protein degradation: An unconventional route to a familiar fate , 1996 .

[23]  K. Bayer,et al.  Kinetic Studies for the Optimization of Recombinant Protein Formation a , 1996, Annals of the New York Academy of Sciences.

[24]  P. Punt,et al.  The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins. , 1997, Gene.

[25]  D. Cavener,et al.  Eukaryotic start and stop translation sites. , 1991, Nucleic acids research.

[26]  R. Takors,et al.  Human Chymotrypsinogen B Production with Pichiapastoris by Integrated Development of Fermentation and Downstream Processing. Part 1. Fermentation , 2001, Biotechnology progress.

[27]  H. Ohi,et al.  Chromosomal DNA patterns and gene stability of Pichia pastoris , 1998, Yeast.

[28]  J. Maat,et al.  Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant Thaumatin in yeast , 1996, Applied Microbiology and Biotechnology.

[29]  M. Penttilä,et al.  Effects of Inactivation and Constitutive Expression of the Unfolded- Protein Response Pathway on Protein Production in the Yeast Saccharomyces cerevisiae , 2003, Applied and Environmental Microbiology.

[30]  N. Borth,et al.  Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. , 2003, Journal of biotechnology.

[31]  L. Alberghina,et al.  In Saccharomyces cerevisiae, protein secretion into the growth medium depends on environmental factors , 1993, Yeast.

[32]  R. Fischer,et al.  Production of carcinoembryonic antigen (CEA) N‐A3 domain in Pichia pastoris by fermentation , 1999, Biotechnology and applied biochemistry.

[33]  W. Stiekema,et al.  Optimization of the expression of equistatin in Pichia pastoris. , 2002, Protein expression and purification.

[34]  R. Parekh,et al.  Multicopy overexpression of bovine pancreatic trypsin inhibitor saturates the protein folding and secretory capacity of Saccharomyces cerevisiae. , 1995, Protein expression and purification.

[35]  K. Wittrup,et al.  Constitutive Overexpression of Secreted Heterologous Proteins Decreases Extractable BiP and Protein Disulfide Isomerase Levels in Saccharomyces cerevisiae , 1995, Biotechnology progress.

[36]  L. Hendershot,et al.  The Unfolding Tale of the Unfolded Protein Response , 2001, Cell.