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Pichia pastoris Cultivation

Pichia pastoris Cultivation

Pichia pastoris is a methylotrophic yeast that provides a unique expression system to produce high levels of recombinant proteins, including enzymes, protease inhibitors, single-chain antibodies and different levels of regulatory proteins. Pasteur yeast is a good research and production culture because it provides a low-cost expression system, similar to E. coli or other prokaryotic cultures, and incorporates post-translational modifications found in eukaryotes.

Lab Scale Cultivation of Pichia pastoris

Pichia pastoris.Figure 1. Pichia pastoris. (From Wikipedia.org)

Table.1 YM Agar Approximate Formula* Per Liter

Medium Component Quantity
Yeast Extract 3.0 g
Malt Extract 3.0 g
Peptone 5.0 g
Dextrose 10.0 g
Agar 20.0 g

Table.2 Pichia pastoris Culture Parameter

Culture parameters Description
Dehydrated Appearance Beige, free-flowing, homogeneous.
Solution 4.1% solution, soluble in purified water upon boiling. Solution is light to medium amber, very slightly opalescent.
Prepared Appearance Light to medium amber, slightly opalescent.
pH Solution at 25°C: pH 6.2 ± 0.2
Culture conditions 28-30℃, aerobic.

Pichia pastoris Strain for Large Scale Production

Currently widely used commercial strains mainly include 5 types, wild-type strains (such as X-33), dystrophic strains (such as GS 115, KM71), protease-deficient strains (such as SMD 1168), glyco-engineered strains (such as SuperMan5) and other strains. At first, one disadvantage of this protein expression system was the excessive glycosylation of the high-density mannose structure. It is worth noting that the engineered P. pastoris has been able to secrete recombinant proteins with consistent human n-strand glycans. The application of transgenic Pasteur yeast broadens the application of microbial systems in antibody expression.

Currently, prokaryotic (bacterial) and eukaryotic (such as yeast, mammalian, insect, and plant) systems have been widely used as host organisms to produce therapeutic proteins and vaccines. During the production process, proteins with a size of more than 100 kDa are usually expressed in eukaryotic cells, while small proteins within 30 kDa are produced in prokaryotic cells. Statistics show that P. pastoris has been used to produce more than 500 proteins, from industrial enzymes to biopharmaceuticals. In addition, many recombinant proteins have been successfully expressed in Pichia pastoris. Table 3 shows several proteins successfully expressed on Pichia pastoris cells.

Table.3 Expression level of several heterologous proteins by Pichia pastoris (g/L) produced in fermenter scale and used for pharmaceutical industry needs.

Type Expression of protein Titer (g/L)
Vaccine Tetanus toxin fragment C 12
Heavy chain botulinum neurotoxin serotype A 1.72*
Antibody fragment A33scFv 4
Anti-HB Fab 0.05
Anti-HbsAg Fab 0.46
Hormone Human parathyroid hormone 0.3
Cytokines Human tumor necrosis factor 10
Bovine interferon gamma 1.0
Ovine interferon tau 0.4
Matrix protein Mouse gelatin 14.8
Human collagen I-III 0.2-06
*Intracellular protein, expression level per gram cell

Why choose Us?

The culture of Pichia pastoris requires specific formulations of growth media for use in protein expression. Creative Biogene offers a selection of microorganism growth media and custom services for your specific application. If you are interested in our microbial anaerobic and aerobic culture platform, please contact us for more details.

References

  1. Vassileva A, et al.; Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter. J Biotechnol. 2001, 88(1):21-35.
  2. Daly R, et al.; Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. Journal of Molecular Recognition. 2005, 18 (2): 119–38.
  3. Julien, C.. Production of humanlike recombinants proteins in Pichia pastoris: From expression vector to fermentation strategy. Bioprocess Technical. 2006, 4(1) pp: 22–31.
For Research Use Only.

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