Announcing...
Pichia 2009 protein expression conference
Visit our Pichia system web site, http://www.pichia.com, for technology information, news, frequently asked questions, licensing information and more.
Pichia platform
The Pichia Expression system is licensed to more than 120 companies in the biotechnology, pharmaceutical, animal health and food industries. A recent improvement, the Pichia GlycoSwitch™ system, adds new genes to Pichia to create strains that glycosylate proteins the same as mammalian cells. These new strains produce proteins that may have increased applications for human therapies.
High yield + high productivity + chemically defined media + product processing similar to mammalian cells + stable production strains + durability = lower protein production costs
Superior Expression
Pichia pastoris is an established industrial yeast that can use methanol as its sole carbon source to produce energy and cellular materials at ultra-high levels (1-5). The first enzyme in the methanol pathway, alcohol oxidase, can make up as much as 35 percent of the soluble protein of the cell (6).
Several genes in Pichia's methanol utilization pathway have been cloned (7-10). Heterologous expression using elements of these genes produces recombinant proteins at levels equivalent to alcohol oxidase when transformed yeast is grown on a methanol feedstock.
High Cell Densities
When Pichia is grown with the proprietary, high-cell-density fermentation technology, unprecedented levels of cell mass per liter of fermentation fluid are produced (4). The system has attained dry-cell-weight densities exceeding 120 gram/liter and continuous fermentation productivities of 10 to 12 grams of recombinant protein/liter/hour.
Controllable Process
The growth medium that feeds Pichia is completely defined (4). It consists of a simple, inexpensive formulation. The carbon source is fed to the fermentor at a rate designed to achieve maximum cell density while maintaining optimal production of foreign protein. This process minimizes any toxic effects the foreign protein might have on the yeast.
Mammalian-like Proteins
As a eukaryotic system, the Pichia Expression system produces mammalian-like proteins. For example, the expression of Hepatitis B surface antigen (I-IBsAg) in Pichia leads to production of particles that are immunoreactive with anti-HBsAg antibodies (11). These particles are similar to Dane particles isolated from the sera of human carriers.
Generations of Stability
Expression of foreign genes is achieved by integration of foreign DNA into the chromosomal DNA of Pichia. The integrated DNA is stable for many generations; all cells can produce the protein (4). In contrast, plasmid-based systems require selective pressure on plasmids to maintain the foreign DNA. Cells that lose the plasmid cannot produce the desired foreign protein.
Durability
The Pichia Expression system requires no special handling. It was developed to withstand the adverse conditions of large scale, continuous fermentors. This feature makes Pichia able to survive unexpected disruptions in the fermentation process.
Maximum Value
High per-cell expression levels combined with high cell-density growth of Pichia translates into greater quantities of recombinant protein per fermentor volume. This reduces production costs by increasing the amount of product per fermentation run.
Protein purification is another cost-saving area. The Pichia Expression system can secrete protein into the medium, so the broth that enters purification contains a higher concentration of the desired protein. Pure protein is recovered with higher yield and lower cost (12).
Patents issued in the United States, Europe, Canada, Japan, Australia and other countries are available on request.
Mammalian- Like Glycosylation - Pichia GlycoSwitch™
In collaboration with Roland Contreras, Ph.D., and colleagues at Ghent University and the Flanders Institute for Biotechnology (VIB) in Belgium, RCT has developed a way to manipulate the Pichia glycosylation system to produce uniform, small ASN-linked glycans on any glycoprotein of interest. These glycans have the same structure as processing intermediates of the mammalian N-glycosylation pathway. This improvement to RCT's Pichia platform is called GlycoSwitch™. If you are interested in evaluating the potential of the GlycoSwitch™ strains and vectors, please contact RCT.
Contact
For licensing:
Andrea Isner, Assistant to David Bramhill, Ph.D.
(520) 748-4400 or (520) 748-0025 (fax)
For kit purchases, technical and custom services:
Invitrogen Corp., (800) 955-6288 or (760) 602-6500 (fax)
References
1. De Schutter, K., Lin,Y., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J., Rouzé, P., Van de Peer, Y., Callewaert, N. (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nature Biotechnology 27, 561-566.
2. Lin, Y., annotator. (2009) Pichia pastoris annotation.
http://bioinformatics.psb.ugent.be/webtools/bogas/annotation/Picpa/666666/chr1-1_0117
3. Cregg, J.M. (2007) Methods in Molecular Biology: Pichia Protocols, Second Edition, Volume 389, Humana Press, Totowa, NJ.
4. Romanos, M.A., Scorer, C.A., and Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
5. Bill, R.M. (2001) Yeast - a panacea for the structure-function analysis. Curr. Genet. 40, 157-171.
6. Lin Cereghino, J., and Cregg, J.M. (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45-66.
7. Higgins, D.R., and Cregg, J.M. (1998) Methods in Molecular Biology: Pichia Protocols, Volume 103, Humana Press, Totowa, NJ.
8. Ilgen, C., Cereghino, J. L., and Cregg, J. M. (2004) Chapter 7: Pichia pastoris. In: Production of recombinant proteins: microbial and eukaryotic expression systems. Gellissen, G. (ed.) Wiley-VCH Verlag, Weinheim, Germany, pp.143-162.
9. Couderc, R., and Baratti, J. (1980) Oxidation of methanol by the yeast Pichia pastoris: purification and properties of alcohol oxidase. Agric. Biol. Chem. 44, 2279-2289.
10. Ellis, S.B., Brust, P.F., Koutz, P.J., Waters, A.F., Harpold, M.M., and Gingeras, T.R. (1985) Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol. Cell. Biol. 5, 1111-1121.
11. Cregg, J.M., Madden, K.R., Barringer, K.J., Thill, G.P., and Stillman, C.A. (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9, 1316-1323.
12. Tschopp, J.F., Brust, P.F., Cregg, J.M., Stillman, C.A., and Gingeras, T.R. (1987a) Expression of the LacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 15, 3859-3876.
13. Shen, S., Sulter, G., Jeffries, T.W., and Cregg, J.M. (1998) A strong nitrogen source Regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene 216, 93-102.
14. Cregg, J.M., Tschopp, J.F., Stillman, C., Siegel, R., Akong, M., Craig, W.S., Buckholz, R.G., Madden, K.R., Kellaris, P.A., Davis, G.R., Smiley, B.L., Cruze, J., Torregrossa, R., Velicelebi, G., and Thill, G.P. (1987) High level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Bio/Technology 5, 479-485.
15. Tschopp, J.F., Sverlow, G., Kosson, R., Craig, W., and Grinna, L. (1987b) High level Secretion of glycosylated invertase in the methylotrophic yeast, Pichia pastoris. Bio/Technology 5, 1305-1308.