Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.

Numerous heterologous proteins have been produced at greater than gram per liter levels in the methylotrophic yeast, Pichia pastoris, using the methanol oxidase promoter. The factors that drastically influence protein production in this system include: copy number of the expression cassette, site and mode of chromosomal integration of the expression cassette, mRNA 5'- and 3'-untranslated regions (UTR), translational start codon (AUG) context, A+T composition of cDNA, transcriptional and translational blocks, nature of secretion signal, endogenous protease activity, host strain physiology, media and growth conditions, and fermentation parameters. All these factors should be considered in designing an optimal production system. The inherent ability of P. pastoris to convert the zymogen (pro-enzyme) form of matrix metalloproteinases (MMP) into active mature forms (which tend to self-degrade, and in some instances also cause damage to cells), largely limits the use of this system for the production of MMP. However, this problem can be partly alleviated by co-expression of tissue inhibitor of MMP (TIMP-1).

[1]  I G Charles,et al.  Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties. , 1991, Vaccine.

[2]  M. Romanos,et al.  High-Level Expression of Tetanus Toxin Fragment C in Pichia Pastoris Strains Containing Multiple Tandem Integrations of the Gene , 1991, Bio/Technology.

[3]  R. C. Dickson,et al.  Construction of strains of Saccharomyces cerevisiae that grow on lactose. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[4]  R. Siegel,et al.  Continuous Production of a Novel Lysozyme via Secretion from the Yeast, Pichia pastoris , 1989, Bio/Technology.

[5]  R. H. Baltz,et al.  Industrial Microorganisms: Basic and Applied Molecular Genetics , 1993 .

[6]  A. Cardin,et al.  Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeast Pichia pastoris. , 1995, Protein expression and purification.

[7]  A. Eisen,et al.  SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. , 1989, The Journal of biological chemistry.

[8]  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.

[9]  J. Tschopp,et al.  High-Level Secretion of Glycosylated Invertase in the Methylotrophic Yeast, Pichia Pastoris , 1987, Bio/Technology.

[10]  S. Wagner,et al.  High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid β-protein precursor , 1992 .

[11]  R. C. Dickson,et al.  Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903. , 1984, Gene.

[12]  L. Matrisian,et al.  Metalloproteinases and their inhibitors in matrix remodeling. , 1990, Trends in genetics : TIG.

[13]  E. Komives,et al.  Expression of highly disulfide-bonded proteins in Pichia pastoris. , 1994, Structure.

[14]  V. Storme,et al.  High–Level Secretion and Very Efficient Isotopic Labeling of Tick Anticoagulant Peptide (TAP) Expressed in the Methylotrophic Yeast, Pichia pastoris , 1994, Bio/Technology.

[15]  R. Potenz,et al.  High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. , 1989, Biochemistry.

[16]  A. Schmaier,et al.  Expression, purification, and characterization of the Kunitz-type proteinase inhibitor domain of the amyloid beta-protein precursor-like protein-2. , 1994, Biochimica et biophysica acta.

[17]  G. Wegner.  Emerging applications of the methylotrophic yeasts. , 1990, FEMS microbiology reviews.

[18]  P. T. Magee,et al.  The Saccharomyces cerevisiae SPR1 gene encodes a sporulation-specific exo-1,3-beta-glucanase which contributes to ascospore thermoresistance , 1993, Journal of bacteriology.

[19]  Z. Werb,et al.  Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix , 1995, Science.

[20]  R. Schmitz,et al.  Generation of Rabbit Monoclonal Antibody Fragments from a Combinatorial Phage Display Library and Their Production in the Yeast Pichia pastoris , 1995, Bio/Technology.

[21]  Michael A. Romanos,et al.  Rapid Selection Using G418 of High Copy Number Transformants of Pichia pastoris for High–level Foreign Gene Expression , 1994, Bio/Technology.

[22]  W. McCombie,et al.  High level expression of heterologous proteins in methylotrophic yeast Pichia pastoris , 1988, Journal of basic microbiology.

[23]  J. Tschopp,et al.  Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation of Pichia pastoris. , 1987, Gene.

[24]  P. Piper,et al.  Overexpression of the Gene for Polyubiquitin in Yeast Confers Increased Secretion of a Human Leucocyte Protease Inhibitor , 1994, Bio/Technology.

[25]  DNA fingerprinting: a new dimension. , 1990, Trends in genetics : TIG.

[26]  R. Cannon,et al.  An exo-beta-(1,3)-glucanase of Candida albicans: purification of the enzyme and molecular cloning of the gene. , 1993, Journal of general microbiology.

[27]  C. Scorer,et al.  Foreign gene expression in yeast: a review , 1992, Yeast.

[28]  J. Cregg,et al.  High–Level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast, Pichia Pastoris , 1987, Bio/Technology.

[29]  C. Scorer,et al.  The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. , 1993, Gene.