# US 8,785,613 B2 · P. pastoris pastoris promoters, and the use thereof to direct expression of proteins in yeast, preferably using a haploid mating strategy > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,785,613 B2 - Document ID: US8785613B2 - Status: Granted - Kind: B2 - Application number: 13/542,851 - Filed: 2012-07-06 - Published: 2012-11-29 - Granted: 2014-07-22 - Earliest claimed: 2006-10-10 - Assignees: Alderbio Holdings LLC - Applicants: James M. Cregg; Ilya I. Tolstorukov - Inventors: James M. Cregg; Ilya I. Tolstorukov - Web page: https://patentgrep.com/patent/US8785613B2 ## CPC - C12N 9/88: Lyases (4.) - C07K 14/39: from yeasts - C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex} - C12N 9/0006: {acting on CH-OH groups as donors (1.1)} - C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases} - C12N 15/81: for yeasts - C12N 15/815: {for yeasts other than Saccharomyces} - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C12N 2830/005: repressible enhancer/promoter combination, e.g. KRAB - C12N 2830/702: yeast - C12Y 402/01011: Phosphopyruvate hydratase (4.2.1.11), i.e. enolase - Y02P 20/52: using catalysts, e.g. selective catalysts - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12Y: ENZYMES - Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS ## Abstract Novel promoters which are derived from P. pastoris pastoris which are inducible or repressible under specific growth conditions are provided. These promoters are useful for regulating the expression of a desired structural gene, e.g., a mammalian polypeptide. Particularly preferred is the use of these novel promoters to regulate gene expression in polyploidal yeast such as diploidal P. pastoris produced by mating or spheroplast fusion. ## Claims ### Claim 1 A nucleic acid construct comprising an isolated P. pastoris glycerol kinase (GUT1) gene promoter sequence comprising the nucleic acid sequence of SEQ ID NO:28 operably linked to a structural gene, wherein the promoter directs the transcription of the structural gene under inducible conditions. ### Claim 2 (depends on 1) The isolated nucleic acid construct of claim 1, wherein said inducible conditions comprise the presence of glycerol or other mixed glycerol feed induction paradigm. ### Claim 3 (depends on 1) The nucleic acid construct of claim 1, wherein the promoter is operably linked to a structural gene other than GUT1. ### Claim 4 (depends on 1) The nucleic acid construct of claim 1, wherein the structural gene is a mammalian gene. ### Claim 5 (depends on 1) The nucleic acid construct of claim 1, which is on a plasmid. ### Claim 6 (depends on 1) The nucleic acid construct of claim 1, wherein said plasmid is an autonomously replicating plasmid or one which integrates into the chromosomal DNA of a yeast cell containing said plasmid. ### Claim 7 (depends on 5) A yeast cell which contains a plasmid according to claim 5. ### Claim 8 (depends on 7) The yeast cell of claim 7, which is a P. pastoris, Saccharomyces, Candida, Yarrowia, Kluyveromyces, Hansenula, or Schizzosaccharomyces yeast cell. ### Claim 9 (depends on 7) The yeast cell of claim 7, which is polyploid. ### Claim 10 (depends on 9) The yeast cell of claim 9, which is a P. pastoris yeast cell. ### Claim 11 (depends on 7) A stable culture containing a yeast cell according to claim 7. ### Claim 12 (depends on 7, 11) A method of producing a protein encoded by a structural gene under the regulatory control of the P. pastoris GUT1 promoter, comprising culturing the yeast cell according to claim 7 or the stable culture according to claim 11 under inducible conditions to produce the protein. ### Claim 13 (depends on 12) The method of claim 12, wherein said protein is a multichain protein. ### Claim 14 (depends on 13) The method of claim 13, wherein said protein is a mammalian protein. ### Claim 15 (depends on 14) The method of claim 14, wherein said mammalian protein is an antibody is specific to a cytokine or growth factor. ### Claim 16 (depends on 15) The method of claim 15, wherein said cytokine is an interferon such as interferon alpha, beta, or gamma, an interleukin such as IL-2, IL-4, IL-6, IL-12, or IL-13, a colony stimulating factor or a tumor necrosis factor and the growth factor is a VEGF. ### Claim 17 (depends on 12) The method of claim 12, wherein said yeast cell is cultured in the presence of an amount of glycerol, or another carbon source or a mixed feed containing glycerol, sufficient to induce expression. ### Claim 18 (depends on 12) The method of claim 12, wherein said yeast cell contains at least one auxotrophic mutation which requires supplementation for growth. ### Claim 19 (depends on 1) A fragment of the GUT1 promoter of claim 1, which when operably linked with a promoter other than the GUT1 promoter renders the promoter glycerol inducible.