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Last Updated: March 29, 2024

Claims for Patent: 9,057,061


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Summary for Patent: 9,057,061
Title:Gene expression technique
Abstract: The present invention provides a method for producing a desired protein (such as a desired heterologous protein) comprising: (a) providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene, such as a third recombinant gene, encoding a desired protein (such as a desired heterologous protein), wherein the first and second chaperones are different; and (b) culturing the host cell in a culture medium to obtain expression of the first, second and third genes.
Inventor(s): Finnis; Christopher John Arthur (Lenton, GB), Sleep; Darrell (West Bridgford, GB), Shuttleworth; Gillian (West Bridgford, GB)
Assignee: NOVOZYMES BIOPHARMA DK A/S (Bagsvaerd, DK)
Application Number:11/722,539
Patent Claims:1. A plasmid comprising a gene encoding a heterologous protein, and as the sole yeast selectable marker, one or more genes encoding an essential chaperone that is essential to the viability of a yeast host cell when the one or more genes encoding the essential chaperone are deleted or inactivated in a yeast host cell and the host cell is inviable in culture and the deficiency cannot be complemented by additions or modifications to the culture medium, wherein the natural location of the one or more genes encoding the essential chaperone are modified to disrupt its function under normal growth conditions wherein the essential chaperone is a yeast chaperone comprising the sequence of a protein encoded by a gene selected from the group consisting of Protein Disulfide Isomerase (PDI) and Protein Disulfide Isomerase 1(PDI1).

2. The plasmid according to claim 1 wherein the essential chaperone is a yeast chaperone.

3. The plasmid in accordance with claim 1, which is a 2.mu.m-family plasmid.

4. The plasmid in accordance with claim 1, which is not a 2.mu.m-family plasmid.

5. The plasmid in accordance with claim 1, wherein the one or more genes encode a first essential chaperone and a second essential chaperone.

6. A plasmid in accordance with claim 5, wherein the second chaperone is selected from the group consisting of (i)-(iv): (i) has a sequence of a fungal chaperone, a yeast chaperone, a mammalian chaperone, or a human chaperone; (ii) comprises the sequence of a protein encoded by any one of Activator of Heat shock protein 90 ATPase 1 (AHA1), Chaperonin Containing TCP-1 1 (CCT2), Chaperonin Containing TCP-1 3 (CCT3), Chaperonin Containing TCP-1 4 (CCT4), Chaperonin Containing TCP-1 5 (CCT5), Chaperonin Containing TCP-1 6 (CCT6), Chaperonin Containing TCP-1 7 (CCT7), Chaperonin Containing TCP-1 8 (CCT8), CyclophiliN Seven suppressor (CNS1), Cyclosporin-sensitive Proline Rotamase 3 (CPRS), Cyclosporin-sensitive Proline Rotamase 6 (CPRE), EPS1, ER Oxidation or Endoplasmic Reticulum Oxidoreductin (ERO1), ER protein Unnecessary for Growth (EUG1), FMO1, High-Copy Hsp90 suppressor (HCH1), Heat Shock Protein 10 (HSP10), Heat Shock Protein 12 (HSP12), Heat Shock Protein 104 (HSP104), Heat Shock Protein 26 (HSP26), Heat Shock Protein 30 (HSP30), Heat Shock Protein 42 (HSP42), Heat Shock Protein 60 (HSP60), Heat Shock Protein 78 (HSP78), Heat Shock Protein 82 (HSP82), DnaJ-like protein of the ER Membrane (JEM1), Mitochondrial DnaJ 1 (MDJ1), Mitochondrial DnaJ 2 (MDJ2), MPD1, MPD2, Protein Disulfide Isomerase (PDI), Protein Disulfide Isomerase 1 (PDI1), PreFolDin (PFD1), COenzyme Q (ABC1), Anti-Prion DnaJ (APJ1), ATP synthase 11 (ATP11), ATP synthase 12 (ATP12), BTf Three (BTT1), Cell Division Cycle 37 (CDC37), Cyclosporin-sensitive Proline Rotamase 7 (CPR7), HSC82, KARyogamy 2 (KAR2), Lumenal Heat Shock Protein 70 (LHS1), Mitochondrial GrpE (MGE1), Translocase of the Inner Membrane 11 (MRS11), Nin1 (One) Binding (NOB1), ExtraCellular Mutant 10(ECM10), Stress-Seventy subfamily A 1 (SSA1), Stress-Seventy subfamily A 2(SSA2), Stress-Seventy subfamily A 2 (SSA3), Stress-Seventy subfamily A 4(SSA4), Stress-Seventy subfamily C 1 (SSC1), SSE2, Suppressor of the Ire1/Lhs1 double mutant (SIL1), Sigma Like Sequence (SLS1), Ubiquitin 4(UBI4), ORM1, ORM2, protein Processing in the ER (PER1), Phosphatase Two C (PTC2), Protein Secretion Enhancer (PSE1), Homologous to Atf/Creb1 (HAC1) or truncated intronless HAC1, Translocase of the Inner Mitochondrial membrane 9 (TIM9), Presequence translocase-Associated Motor 18 (PAM18) or Tailless Complex Polypeptide (TCP1) or a variant or fragment of any one of these; (iii) is protein disulphide isomerase; and (iv) is Orm2p or a variant or fragment thereof.

7. The plasmid in accordance with claim 5, wherein the plasmid comprises a third recombinant gene, which third recombinant gene encodes a desired heterologous protein.

8. The plasmid in accordance with claim 5, wherein the plasmid is a disintegration vector.

9. A host cell comprising the plasmid of claim 1, the plasmid further comprising a recombinant gene encoding a desired heterologous protein, wherein: (i) the essential chaperone is not HSP10; and (ii) the desired heterologous protein: (a) comprises a leader sequence; and/or (b) is not identical in sequence to a protein that is encoded by one or more other genes that occur naturally in the host cell; and/or (c) is a therapeutically active protein; and/or (d) is a diagnostically, industrially, domestically or nutritionally useful protein; and/or (e) is a protein selected from the group consisting of the sequence of albumin, a monoclonal antibody, an etoposide, a serum protein, antistasin, a tick anticoagulant peptide, transferrin, lactoferrin, endostatin, angiostatin, collagens, immunoglobulins, immunoglobulin-based molecules, a Kunitz domain protein, interferons, interleukins, IL10,IL11, IL2, interferon .alpha. species and subspecies, interferon .beta. species and sub-species, interferon .gamma. species and sub-species, leptin, CNTF, CNTF.sub.Ax15, IL1-receptor antagonist, erythropoietin (EPO), EPO mimics, thrombopoietin, TPO mimics, prosaptide, cyanovirin-N, 5-helix, T20peptide, T1249 peptide, HIV gp41, HIV gp120, urokinase, prourokinase, tPA, hirudin, platelet derived growth factor, parathyroid hormone, proinsulin, insulin, glucagon, glucagon-like peptides, insulin-like growth factor, calcitonin, growth hormone, transforming growth factor .beta., tumor necrosis factor, G-CSF, GM-CSF, M-CSF, FGF, coagulation factors , plasminogen, fibrinogen, thrombin, pre-thrombin, pro-thrombin, von Willebrand's factor, .alpha..sub.1-antitrypsin, plasminogen activators, Factor VII, Factor VIII, Factor IX, Factor X and Factor XIII, nerve growth factor, LACI, platelet-derived endothelial cell growth factor (PD-ECGF), glucose oxidase, serum cholinesterase, aprotinin, amyloid precursor protein, inter-alpha trypsin inhibitor, antithrombin III, apo-lipoprotein species, Protein C, Protein S, a metabolite, an antibiotic, a variant of any of the above, and functional fragment of any of the above.

10. The host cell in accordance with claim 9, wherein the essential chaperone is a yeast chaperone.

11. The host cell in accordance with claim 9, wherein, in the absence of the plasmid, the host cell is inviable.

12. The host cell according to claim 11 wherein, in the absence of the plasmid, the host cell is inviable and cannot be made viable by growing the host cell in a growth medium and making nutrient additions or modifications to that growth medium, and preferably cannot be made viable by growing the host cell in a growth medium and making any additions or modifications to that growth medium.

13. A host cell comprising the plasmid of claim 5.

14. The host cell according to claim 13 wherein, in the absence of the plasmid, the host cell does not produce the chaperone.

15. A method for producing a desired protein comprising: (a) providing the host cell in accordance with claim 13 comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene, such a third recombinant gene, encoding a desired protein, wherein the first and second chaperones are different; and (b) culturing the host cell in a culture medium to obtain expression of the first, second and third genes.

16. A method for producing a desired recombinant protein comprising the steps of: providing a host cell comprising the plasmid of claim 1, and a gene that encodes a desired protein; culturing the host cell in a culture medium under conditions that allow the expression of the essential chaperone and the desired protein.

17. The method according to claim 16, wherein the plasmid further comprises a recombinant gene encoding a desired heterologous protein wherein: (i) the essential chaperone is not HSP10; and (ii) the desired heterologous protein: (a) comprises a leader sequence; and/or (b) is not identical in sequence to a protein that is encoded by one or more other genes that occur naturally in the host cell; and/or (c) is a therapeutically active protein; and/or (d) is a diagnostically, industrially, domestically or nutritionally useful protein; and/or (e) is a protein comprising the sequence of albumin, a monoclonal antibody, an etoposide, a serum protein, antistasin, a tick anticoagulant peptide, transferrin, lactoferrin, endostatin, angiostatin, collagens, immunoglobulins, immunoglobulin-based molecules, a Kunitz domain protein, interferons, interleukins, IL10,IL11, IL2, interferon a species and sub-species, interferon .beta.species and sub-species, interferon .gamma. species and sub-species, leptin, CNTF, CNTF.sub.Ax15, IL1-receptor antagonist, erythropoietin (EPO) and EPO mimics, thrombopoietin (TPO) and TPO mimics, prosaptide, cyanovirin-N, 5-helix, T20peptide, T1249 peptide, HIV gp41, HIV gp120, urokinase, prourokinase, tPA, hirudin, platelet derived growth factor, parathyroid hormone, proinsulin, insulin, glucagon, glucagon-like peptides, insulin-like growth factor, calcitonin, growth hormone, transforming growth factor .beta., tumor necrosis factor, G-CSF, GM-CSF, M-CSF, FGF, coagulation factors in both pre and active forms, plasminogen, fibrinogen, thrombin, pre-thrombin, pro-thrombin, von Willebrand's factor, a.sub.1-antitrypsin, plasminogen activators, Factor VII, Factor VIII, Factor IX, Factor X and Factor XIII, nerve growth factor, LACI, platelet-derived endothelial cell growth factor (PD-ECGF), glucose oxidase, serum cholinesterase, aprotinin, amyloid precursor protein, inter-alpha trypsin inhibitor, antithrombin III, apo-lipoprotein species, Protein C, Protein S, a metabolite, an antibiotic, or a variant or functional fragment of any of the above.

18. A method for producing a desired protein comprising the steps of: (a) providing the host cell in accordance with claim 9; and (b) culturing the host cell in a culture medium under conditions that allow the expression of the essential chaperone and the desired protein.

19. The host cell in accordance with claim 9, wherein the host cell is a bacterial or yeast host cell.

20. The host cell in accordance with claim 9, wherein the host cell is a yeast cell selected from the group consisting of Saccharomyces, Kluyveromyces, Arxula, Yarrowia, Candida, Schizosaccharomyces, Debaryomyces, Xanthophyllomyces, Geotrichum, Ashbya, Hortaea, Schwanniomyces, Trichosporon, Xanthophyllomyces, Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris, Pichia membranaefaciens, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Kluyveromyces drosphilarum, Pichia methanolica, Hansenula polymorpha, Pichia augusta, Arxula adeninivorans, Yarrowia lipolytica, Candida boidinii Candida utilis and Schizosaccharomyces pombe.

21. The host cell in accordance with claim 20, wherein the plasmid is a 2.mu.m-family plasmid and wherein: (a) the plasmid is based on pSR1, pSB3 or pSB4 and the host cell is Zygosaccharomyces rouxii; (b) the plasmid is based on pSB1 or pSB2 and the host cell is Zygosaccharomyces bailli; (c) the plasmid is based on pSM1 and the host cell is Zygosaccharomyces Fermentati; (d) the plasmid is based on pKD1 and the host cell is Kluyveromyces Drosophilarum; (e) the plasmid is based on pPM1 and the host cell is Pichia membranaefaciens; or (f) the plasmid is based on the 2.mu.m plasmid and the host cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis.

22. The host cell in accordance with claim 19, in which the plasmid is based on the 2.mu.m plasmid and the host cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis.

Details for Patent 9,057,061

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Bayer Healthcare Pharmaceuticals Inc. TRASYLOL aprotinin Injection 020304 12/29/1993 ⤷  Try a Trial 2023-12-23
Microbix Biosystems Inc. KINLYTIC urokinase For Injection 021846 01/16/1978 ⤷  Try a Trial 2023-12-23
Nps Pharmaceuticals, Inc. NATPARA parathyroid hormone For Injection 125511 01/23/2015 ⤷  Try a Trial 2023-12-23
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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