Foreign gene expression in yeast: a review.

The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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Heterologous protein production using the Pichia pastoris expression system.

A process for purifying an antibody is provided. In this process, a mixture containing the antibody and contaminant is subjected to low pH hydrophobic interaction chromatography (LPHIC) optionally at low salt concentration. The antibody is eluted from the column in the fraction which does not bind thereto. This process can be preceded and followed by other purification steps.

Low pH hydrophobic interaction chromatography for antibody purification

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

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.

/pdf/operational-strategies-monitoring-and-control-of-232e2502sg.pdf

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

Insulin-like growth factor-1 (IGF-1), a naturally occurring, relatively short, single chain polypeptide, is prepared by growing methylotrophic yeast transformants containing in their genome at least one copy of DNA encoding IGF-1, in operational linkage with DNA encoding a signal sequence, which is effective for directing secretion of proteins from the host cells and which also includes the proteolytic processing site lys-arg and may include one or more glu-ala sequences. In preferred embodiments the signal sequence is the S. cerevisiae alpha mating factor pre-pro sequence. Expression of both the DNA encoding IGF-1 and the pre-pro signal sequence is regulated by a promoter region derived from a methanol responsive gene of a methylotrophic yeast. DNA constructs and recombinant methylotrophic yeast strains used for the expression and secretion of IGF-1 are also provided. For preferred embodiments, protease deficient Pichia pastoris strains are provided.

Production of insulin-like growth factor-1 in methylotrophic yeast cells

Method for the recovery and purification of intact, correctly-folded, monomeric insulin-like growth factor-1 peptide from large volumes of IGF-1-containing medium are described, comprising a series of adsorption-desorption steps employing a combination of cation exchange and hydrophobic interaction adsorbents. Product IGF-1 peptide is highly purified and suitable for use in a variety of clinical applications.

Method for the purification of intact, correctly-folded insulin-like growth factor-1

The invention provides a method of increasing the production of a recombinant gene product from a culture of a recombinant methylotrophic yeast host, wherein said product is made by expression from a recombinant gene sequence operably associated with a methanol-responsive expression control element. In the method of the invention, the methylotrophic yeast host is first cultured on a medium with a high concentration of multi-carbon, carbon-source nutriment, such as glycerol, but with little or no methanol, in order to increase the density of the host cells with little or no expression of the recombinant gene product. When the host cells have achieved a suitable density in the culture medium, the culture is subjected to a phase during which the concentration of multi-carbon, carbon-source nutriments is maintained sufficiently low that the methanol-responsive control element controlling expression of the recombinant gene encoding the desired product is derepressed. Finally, the culture is subjected to a phase of high production of the recombinant gene product by increasing the concentration of methanol while maintaining the concentration of multi-carbon, carbon-source nutriments at a low level. The invention is illustrated with production, using Pichia pastoris, of bovine lysozyme c2, human lysozyme, human epidermal growth factors (1-52) and (1-48), and human superoxide dismutase.

Mixed feed recombinant yeast fermentation

The present invention provides compositions and methods for the production of the enzyme superoxide dismutase, and particularly human superoxide dismutase, by culturing P. pastoris that has been transformed to express the enzyme.

Production of superoxide dismutase in pichia pastoris yeast cells

L'invention concerne des compositions et des procedes permettant la production de l'enzyme peroxyde dismutase, et notamment de peroxyde dismutase humaine, par culture de P. pastoris ayant ete transforme pour exprimer l'enzyme.

Gregory Clyde Holtz

Papers

Production of insulin-like growth factor-1 in methylotrophic yeast cells

Method for the purification of intact, correctly-folded insulin-like growth factor-1

Mixed feed recombinant yeast fermentation

Production of superoxide dismutase in pichia pastoris yeast cells

Production de peroxyde dismutase dans des cellules de levure de pichia pastoris