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.

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.

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

Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris.

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

The isolation and characterization of genes involved in proteolytic processing in species of the genus Pichia is described. The availability of such genes has enabled the generation of strains of Pichia which are deficient in proteolytic activity, which strains are useful as hosts for the expression of proteolytically sensitive recombinant products. The isolation and characterization of additional genes from species of the genus Pichia is also described, as well as uses therefore.

Genes which influence pichia proteolytic activity, and uses therefor

PROBLEM TO BE SOLVED: To provide strains of the genus Pichia which are deficient in proteolytic activity, and to provide a means for generating such strains. SOLUTION: The isolation and characterization of genes involved in proteolytic processing in species of the genus Pichia and the availability of such genes enable the generation of the strains of Pichia which are deficient in proteolytic activity and useful as hosts for the expression of proteolytically sensitive recombinant products. The isolation and characterization of additional genes from species of the genus Pichia are also provided, as well as uses thereof. COPYRIGHT: (C)2003,JPO

Gene influencing pichia proteolytic activity and use thereof

On prepare le facteur-1 de croissance similaire a l'insuline (IGF1-), un polypeptide naturel, a chaine unique et relativement court, en provoquant la croissance de transformateurs de levure methylotrophique contenant dans leur genome au moins une copie de l'ADN codant IGF-1, en liaison operationnelle avec l'ADN codant une sequence de signaux, qui est efficace pour diriger la secretion de proteines a partir des cellules hotes et qui comprend egalement le site de traitement proteolytique lys-arg et peut comprendre une ou plusieurs sequences glu-ala. Dans les aspects preferes, la sequence de signaux est representee par la sequence pre-pro du facteur d'accouplement alpha de S. cerevisiae. L'expression de l'ADN codant IGF-1 et de la sequence de signaux pre-pro est regulee par une region promotrice provenant d'un gene sensible au methanol d'une levure methylotrophique. L'invention decrit egalement les structures d'ADN et les souches de levure methylotrophique recombinantes utilisees pour l'expression et les secretions de IGF-1. Des aspects preferes mettent en application des souches de Pichia pastoris deficientes en proteases.

Production du facteur-1 de croissance similaire a l'insuline dans des cellules de levure methylotrophique

L'invention concerne l'isolement et la caracterisation de genes impliques dans le traitement proteolytique dans des especes du genre Pichia. La disponibilite de tels genes a permis de produire des souches de Pichia qui sont deficientes en activite proteolytique et qui sont utiles en tant qu'hotes pour l'expression de produits recombinants a sensibilite proteolytique. L'isolement et la caracterisation de genes additionnels a partir d'especes du genre Pichia sont egalement decrits, ainsi que leurs utilisatons.

Bradley Drake Howard

Papers

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

Genes which influence pichia proteolytic activity, and uses therefor

Gene influencing pichia proteolytic activity and use thereof

Production du facteur-1 de croissance similaire a l'insuline dans des cellules de levure methylotrophique

Genes influançant l'activite proteolytique de pichia, et leurs utilisations