Production of recombinant proteins by microbes and higher organisms.

Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris.

Recombinant protein expression for therapeutic applications.

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

In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Production of these proteins has a remarkable demand in the market. Escherichia coli offers a means for the rapid and economical production of recombinant proteins. These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically therapeutic proteins such as insulin and bovine growth hormone. These demands have driven the development of a variety of strategies for achieving high-level expression of protein, particularly involving several aspects such as expression vectors design, gene dosage, promoter strength (transcriptional regulation), mRNA stability, translation initiation and termination (translational regulation), host design considerations, codon usage, and fermentation factors available for manipulating the expression conditions, which are the major challenges is obtaining the high yield of protein at low cost.

Strategies for efficient production of heterologous proteins in Escherichia coli.

A continuous fermentation process has been developed in Pichia pastoris (P. pastoris) with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in order to produce large quantities of recombinant human chitinase (rh-chitinase) for preclinical studies as a potential high-dose antifungal drug. Expression levels of about 200 to 400 mg/L have been demonstrated in fed-batch fermentations using strains with either the traditional methanol-inducible or the constitutive GAP promoter. Proteolytic degradation of the enzyme was typically seen in fed-batch fermentations. Continuous production of the enzyme by P. pastoris with the GAP promoter was demonstrated in a 1.5-L working volume fermentor using either glucose or glycerol as the carbon source. The fermentation could be extended for >1 month with a steady-state protein concentration of approximately 300 mg/L. Cell densities were >400 g/L wet cell weight (WCW) (approximately 100 g/L dry cell weight [DCW]) at a dilution rate (D) of 0.83 day(-1) or 1.2 volume exchanges per day (VVD). No proteolytic degradation of the enzyme was seen in the continuous fermentation mode.

B. M. Schilling

Papers

High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system.