Chemistry: molecular biology and microbiology – Vector – per se
Patent
1994-09-09
1997-10-14
Elliott, George G.
Chemistry: molecular biology and microbiology
Vector, per se
536 231, 536 234, 435 697, 435 699, 435 711, 435255, 4353201, C07H 2104, C12N 1563, C12N 1500, C12P 2100
Patent
active
056771720
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention comprises a method, by which useful proteins are secreted through the secretory route of yeast while being posttranslationally modified. The production and secretion is achieved by transformation of yeast cells with a recombinant DNA vector containing the isolated or synthesized HSP150 gene, or a fragment or derivative thereof, of Saccharomyces cerevisiae or another yeast, joined to a gene encoding a foreign protein. The fusion gene is expressed under the control of the HSP150 control elements or other control elements.
DESCRIPTION OF THE PRIOR ART
The yeast cells offer an alternative for the prokaryotes for the production of heterologous proteins by recombinant DNA-technology mainly because they can secrete proteins via the secretory pathway to the exterior of the plasma membrane, either to the cell wall or to the growth medium (Terkamp-Olson and Valenzuela, Curt. Opinion Biotechnol. 1:28-35, 1990). The secretory pathway of yeast consists of the endoplasmic reticulum (ER), Golgi apparatus and secretory vesicles, and it operates similarly as the mammalian secretory pathway. A nascent or newly synthesized polypeptide crosses the ER membrane due to its signal sequence, and is translocated to the lumen of the ER. Vesicles bud off from the ER membrane and fuse with the Golgi membrane and thus transport secretory proteins to the Golgi. The Golgi consists of many membrane-bound compartments through which the secretory proteins pass by vesicular traffic. The vesicles which bud off from the last Golgi subcompartment, fuse with the plasma membrane, and thus the soluble contents of the secretory vesicles are delivered to the cell wall, or to the growth medium. The enzymes resident in the compartments of the secretory route modify secretory proteins with glycans, proteolytic cleavages, disulfide bonds, fatty acylation, oligomerization etc. (Schekman, Annu Rev. Cell Biol. 1:115-143, 1985). These modifications occur similarly, though not identically, in yeast and mammalian cells. For instance, the glycans are different in these two organisms (Ballou, The Molecular Biology of the Yeast Saccharomyces, Strathern, Jones and Broach (eds.) Cold Spring Harbor Laboratory, Cold Spring Harbor, pp. 335-360, 1992). The modifications yield a protein molecule whose conformation is correct or more correct than that of a protein produced in prokaryotic cells (Curtis et al., PNAS 88:5809-5813, 1991). Many of the desired heterologous proteins are secretory proteins. Mammalian proteins expressed in yeast are expected to be modified and folded authentically, and to be secreted to the growth medium. Secretion is desired, since purification of the product from the medium is far more easy than from cell lysates.
Diffusion Across the Cell Wall
The number of known proteins which are secreted from S. cerevisiae to the cell wall or growth medium is only about 15. Most of them remain intercalated within the cell wall (de Nobel and Barnett, Yeast 7:313-132, 1991). .alpha.-Factor (Julius et al., Cell 36:309-318, 1984), Barl protease.degree. (MacKay et al., PNAS 85:55-59, 188), killer toxin and exo-.beta.-1-3-glucanase (Klebl and Tanner, J. Bacteriol. 171:6259-6264, 1989) cross the cell wall and diffuse to the growth medium. The small size of .alpha.-factor and killer toxin may explain their facile diffusion across the cell wall, whereas the rules by which other proteins are or are not retained within the cell wall are not known.
Glycosylation
Almost all known secretory proteins of S cerevisiae are N-glycosylated during secretion. N-glycan may consist of as many as 200 mannose residues. Yeast-specific N-glycosylation may alter the biological activity of a mannnalian protein, and since N-glycans are immunogenic, proteins which carry them cannot be used as therapeutic agents (Kukuruzinska et al., Annu Rev. Biochem. 56:914-944, 1987).
Regulation
Most of the proteins secreted to the exterior of the plasma membrane of S. cerevisiae are not expressed constitutively, but regulated by nutritional conditions the matin
REFERENCES:
patent: 4797359 (1989-01-01), Finkelstein
patent: 4914026 (1990-04-01), Brake et al.
Nevalainen, "Membrane Traffic in Mitotic and Interphase Yeast Cells", Doctoral thesis, Univ. of Helsinki, pp. 1-61 (1991).
Makarow et al, Proc. Natl. Acad. Sci. USA, vol. 83, pp. 8117-8121 (Nov. 1986).
Duran-Torres et al, "A Secretory Heat Shock Protein of Saccharomyces cerevisiae", pp. 1-15.
Nevalainen et al, Eur. J. Biochem., vol. 178, pp. 39-46 (1988).
Nevalainen et al, Eur. J. Biochem., vol. 184, pp. 165-172 (1989).
Makarow et al, The Journal of Cell Biology, vol. 104, pp. 67-75 (Jan. 1987).
Nevalainen et al, "Uptake of Endocytic Markers into Mitotic Yeast Cells", Univ. of Helsinki, pp. 1-6 (1991).
Ruohola, Doctoral thesis, "Biochemical and Genetic . . . in Yeast", Univ. of Helsinki, Abstract (1989).
Brake et al, Proc. Natl. Acad. Sci. USA, vol. 81, pp.4642-4646 (Aug. 1984).
Lupashin et al, Dokl. Akad. Nauk SSSR, 317(5), pp. 1257-1260 (1991).
Simonen et al, "Journal of Biological Chemistry" vol. 269, No. pp. 1-7 (1994).
Jamsa et al "Yeast" vol. 10, pp. 355-370 (1994).
Russo et al, "Proc. Natl. Acad. Sci. USA" vol. 89, pp. 3671-3675 (1992).
Elliott George G.
Wang Andrew
LandOfFree
Method for production of proteins in yeast does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for production of proteins in yeast, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for production of proteins in yeast will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1554441