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Last Updated: September 27, 2020

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Claims for Patent: 8,252,551

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Summary for Patent: 8,252,551
Title:2-micron family plasmid and use thereof
Abstract: The present invention provides a 2 .mu.m-family plasmid comprising a polynucleotide sequence insertion, deletion and/or substitution between the first base after the last functional codon of at least one of either a REP2 gene or an FLP gene and the last base before the FRT site in an inverted repeat adjacent to said gene.
Inventor(s): Sleep; Darrell (West Bridgford, GB), Finnis; Christopher John Arthur (Lenton, GB)
Assignee: Novozymes Biopharma DK A/S (Bagsvaerd, DK)
Application Number:10/584,486
Patent Claims:1. A 2 .mu.m-family plasmid comprising a polynucleotide sequence insertion, deletion and/or substitution between a first base after a last functional codon of at least one of either an REP2 gene or an FLP gene and a last base before an FRT site in an inverted repeat adjacent to said gene.

2. The 2 .mu.m-family plasmid of claim 1 wherein, other than the polynucleotide sequence insertion, deletion and/or substitution, the FLP gene and/or the REP2 gene has the sequence of an FLP gene and/or an REP2 gene from a naturally occurring 2 .mu.m-family plasmid.

3. The 2 .mu.m-family plasmid of claim 1, wherein the plasmid comprises pSR1, pSB3 or pSB4 from Zygosaccharomyces rouxii, pSB 1 from Zygosaccharomyces bailli, pSB2 from Zygosaccharomyces balli, pSM1 from Zygosaccharomyces fermentati, pKD1 from Kluyveromyces drosophilarum, pPM1 from Pichia membranaefaciens, or the 2 .mu.m plasmid from Saccharomyces cerevisiae.

4. The 2 .mu.m-family plasmid of claim 2 wherein the sequence of the inverted repeat adjacent to said FLP and/or REP2 gene is from the sequence of the corresponding inverted repeat in the same naturally occurring 2 .mu.m-family plasmid as the sequence from which the gene is from.

5. The 2 .mu.m-family plasmid of claim 2 wherein the naturally occurring 2 .mu.m-family plasmid is the 2 .mu.m plasmid as from Saccharomyces cerevisiae.

6. The 2 .mu.m-family plasmid of claim 5 wherein the polynucleotide sequence insertion, deletion and/or substitution occurs at a position between a first base of codon 59 of the REP2 gene and the last base before the FRT site in the adjacent inverted repeat.

7. The 2 .mu.m-family plasmid of claim 5 wherein, other than the polynucleotide sequence insertion, deletion and/or substitution, the sequence of the REP2 gene and the adjacent inverted repeat comprises the nucleotides of SEQ ID NO: 1, or a nucleotide sequence 95% identical to SEQ ID NO:1.

8. The 2 .mu.m-family plasmid of claim 1 wherein polynucleotide sequence insertion, deletion and/or substitution occurs at a position between a first base of the inverted repeat and a last base before the FRT site.

9. The 2 .mu.m-family plasmid of claim 1 wherein the polynucleotide sequence insertion, deletion and/or substitution occurs between a first base after the end of the REP2 coding sequence and the last base before the FRT site.

10. The 2 .mu.m-family plasmid of claim 1 wherein, other than the polynucleotide sequence insertion, deletion and/or substitution, the inverted repeat that follows the REP2 coding sequence has a sequence from a corresponding region of the 2 .mu.m plasmid from Saccharomyces cerevisiae.

11. The 2 .mu.m-family plasmid of claim 5 wherein the polynucleotide sequence insertion, deletion and/or substitution occurs at a position between a first base of codon 344 of the FLP gene and the last base before the FRT site in the adjacent inverted repeat.

12. The 2 .mu.m-family plasmid of claim 5 wherein, other than the polynucleotide sequence insertion, deletion and/or substitution, the sequence of the FLP coding sequence and the adjacent inverted repeat comprises the nucleotides of SEQ ID NO: 2, or a nucleotide sequence 95% identical to SEQ ID NO:2.

13. The 2 .mu.m-family plasmid of claim 11 wherein the polynucleotide sequence insertion, deletion and/or substitution occurs at a position between a first base of the inverted repeat and the last base before the FRT site.

14. The 2 .mu.m-family plasmid of claim 13 wherein the polynucleotide sequence insertion, deletion and/or substitution occurs at a position between a first base after the end of the FLP coding sequence and the last base before the FRT site.

15. The 2 .mu.m-family plasmid of claim 14 wherein the polynucleotide sequence insertion, deletion and/or substitution occurs at a first base after the end of the FLP coding sequence.

16. The 2 .mu.m-family plasmid of claim 11 wherein, other than the polynucleotide sequence insertion, deletion and/or substitution, the inverted repeat that follows the FLP gene has a sequence from a corresponding region of the 2 .mu.m plasmid from Saccharomyces cerevisiae.

17. The 2 .mu.m-family plasmid of claim 1 comprising polynucleotide sequence insertions, deletions and/or substitutions between a first base after the last functional codons of both of the REP2 gene and the FLP gene and a last base before the FRT sites in the inverted repeats adjacent to each of said genes, which polynucleotide sequence insertions, deletions and/or substitutions can be the same or different.

18. The 2 .mu.m-family plasmid of claim 1, comprising a polynucleotide sequence insertion, deletion and/or substitution which is not between the first base and the last base.

19. The 2 .mu.m-family plasmid of claim 18 wherein the polynucleotide sequence insertion, deletion and/or substitution occurs within an untranscribed region around an ARS sequence.

20. The 2 .mu.m-family plasmid of claim 1 wherein the, or at least one, polynucleotide sequence insertion, deletion and/or substitution is a polynucleotide sequence insertion.

21. The 2 .mu.m-family plasmid of claim 20 in which the polynucleotide sequence insertion encodes an open reading frame.

22. The 2 .mu.m-family plasmid of claim 21 in which the open reading frame encodes a non-2 .mu.m-family plasmid protein.

23. The 2 .mu.m-family plasmid of claim 22 in which the non-2 .mu.m-family plasmid protein comprises the sequence of a protein involved in protein folding, or which has chaperone activity or is involved in the unfolded protein response, albumin, a monoclonal antibody, an etoposide, a serum protein, antistasin, a tick anticoagulant peptide, transferrin, lactoferrin, endostatin, angiostatin, collagens, immunoglobulins or immunoglobulin-based molecules or fragments of either, a Kunitz domain protein, interferons, interleukins, IL 10, IL 11, IL2, interferon .alpha. species and sub-species, interferon .beta. species and sub-species, interferon .gamma. species and subspecies, leptin, CNTF, CNTF.sub.Ax15, IL 1-receptor antagonist, erythropoietin (EPO) and EPO mimics, thrombopoietin (TPO) and TPO mimics, prosaptide, cyanovirin-N, 5-helix, T20 peptide, T1249 peptide, HIV gp4I, 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., tumour necrosis factor, G-CSF, GM-CSF, M-CSF, FGF, coagulation factors in both pre and active forms, including but not limited to 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, or Protein S.

24. The 2 .mu.m-family plasmid of claim 23 in which the 2 .mu.m-family plasmid protein comprises the sequence of albumin.

25. The 2 .mu.m-family plasmid of claim 23 in which the non-2 .mu.m-family plasmid protein comprises the sequence of transferrin.

26. The 2 .mu.m-family plasmid of claim 23 in which the non-2 .mu.m-family plasmid protein comprises the sequence of lactoferrin.

27. The 2 .mu.m-family plasmid of claim 23 in which the non-2 .mu.m-family plasmid protein comprises the sequence of Fc.

28. The 2 .mu.m-family plasmid of claim 23 in which the non-2 .mu.m-family plasmid protein comprises the sequence of a protein involved in protein folding, or which has chaperone activity or is involved in the unfolded protein response as encoded by anyone of AHA1, CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNS1, CPR3, CPR6, EPS1, ERO1, EUG1, FMO1, HCH1, HSP10, HSP12, HSP104, HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, JEM1, MDJ1, MDJ2, MPD1, MPD2, PDI1, PFD1, ABC1, APJ1, ATP11, ATP12, BTT1, CDC37, CPR7, HSC82, KAR2, LHS1, MGE1, MRS11, NOB1, ECM10, SSA1, SSA2, SSA3, SSA4, SSC1, SSE2, SIL1, SLS1, UBI4, ORM1, ORM2, PER1, PTC2, PSE1 and HAC1 or a truncated intronless HAC1.

29. The 2 .mu.m-family plasmid of claim 23 in which the chaperone is protein disulphide isomerase (PDI), or is a protein encoded by ORM2, SSA1 or PSE1.

30. The 2 .mu.m-family plasmid of claim 22 in which the non-2 .mu.m-family plasmid protein comprises a secretion leader sequence.

31. The 2 .mu.m-family plasmid of claim 22 in which the non-2 .mu.m-family plasmid protein comprises the sequence of a bacterial selectable marker and/or a yeast selectable marker.

32. The 2 .mu.m-family plasmid of claim 31 in which the bacterial selectable marker is a .beta.-lactamase gene and/or the yeast selectable marker is a LEU2 selectable marker.

33. The 2 .mu.m-family plasmid according to claim 1, which plasmid comprises (i) a heterologous sequence encoding a non-2 .mu.m-family plasmid protein; (ii) a heterologous sequence encoding a protein comprising the sequence of a protein involved in protein folding, a chaperone or a protein involved in the unfolded protein response; and (iii) a heterologous sequence encoding a protein comprising the sequence of a selectable marker; wherein at least one of the heterologous sequences occurs between the first base after the last functional codon of at least one of either the REP2 gene or the FLP gene and the last base before the FRT site in an inverted repeat adjacent to the gene.

34. A method of preparing a plasmid as defined by claim 1 comprising: (a) providing a plasmid comprising the sequence of a REP2 gene and the inverted repeat that follows the REP2 gene, or a FLP gene and the inverted repeat that follows the FLP gene, in each case the inverted repeat comprising an FRT site; (b) providing a polynucleotide sequence and inserting the polynucleotide sequence into the plasmid of claim 1 between the first base after the last functional codon of at least one of either the REP2 gene or the FLP gene and the last base before the FRT site in an inverted repeat adjacent to the gene; and/or (c) deleting some or all of the nucleotide bases between the first base after the last functional codon of at least one of either the REP2 gene or the FLP gene and the last base before the FRT site in an inverted repeat adjacent to the gene of claim 1; and/or (d) substituting some or all of the nucleotide bases between the first base after the last functional codon of at least one of either the REP2 gene or the FLP gene and the last base before the FRT site in an inverted repeat adjacent to the gene with alternative nucleotide bases.

35. A plasmid obtainable by the method of claim 34.

36. A host cell comprising a plasmid as defined by claim 1.

37. A host cell according to claim 36 which is a yeast cell.

38. A host cell according to claim 36 in which the plasmid is stable as a multicopy plasmid.

39. A host cell according to claim 38 in which the plasmid comprises a polynucleotide sequence insertion, deletion and/or substitution between a first base after a last functional codon of at least one of either an REP2 gene or an FLP gene and a last base before an FRT site in an inverted repeat adjacent to said gene.

40. A host cell according to claim 38 in which, if the plasmid contains, or is modified to contain, a selectable marker then stability, as measured by the loss of the marker, is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% after 5 generations.

41. A method of producing a protein comprising the steps of-- (a) providing a plasmid as defined by claim 1; (b) providing a suitable host cell; (c) transforming the host cell with the plasmid; and (d) culturing the transformed host cell in a culture medium; (e) thereby to produce the protein.

42. A method of producing a protein comprising the steps of providing a host cell as defined by claim 36 which host cell comprises a plasmid comprising a polynucleotide sequence insertion, deletion and/or substitution between the first base after the last functional codon of at least one of either a REP2 gene or an FLP gene and the last base before the FRT site in an inverted repeat adjacent to said gene as and culturing the host cell in a culture medium thereby to produce the protein.

43. A method according to claim 41 further comprising the step of isolating the thus produced protein from the cultured host cell or the culture medium.

44. A method according to claim 43 further comprising the step of purifying the thus isolated protein.

45. A method according to claim 44 further comprising the step of formulating the thus purified protein with a carrier or diluent, and optionally presenting the thus formulated protein in a unit form.

46. A method according to claim 44 further comprising the step of formulating the purified protein with a pharmaceutically acceptable carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form.

47. The 2 .mu.m-family plasmid of claim 11, wherein the polynucleotide sequence insertion, deletion and/or substitution occurs at an HgaI site or an FspI site within the inverted repeat.

48. The 2 .mu.m-family plasmid of claim 1, wherein the plasmid comprises a heterologous sequence encoding protein disulphide isomerase.

49. The 2 .mu.m-family plasmid of claim 1, wherein the plasmid comprises a heterologous sequence encoding a protein of interest.

50. The 2 .mu.m-family plasmid of claim 22 in which the non-2 .mu.m-family plasmid protein comprises immunoglobulin-based molecules or fragments thereof selected from the group consisting of dAb, Fab', F(ab')2, scAb, scFv andor scFv.

51. A 2 .mu.m-family plasmid comprising a polynucleotide sequence insertion between a first base after a last functional codon of at least one of either an REP2 gene or an FLP gene and a last base before an FRT site in an inverted repeat adjacent to said gene, wherein the polynucleotide sequence insertion encodes an open reading frame which encodes a non-2 .mu.m-family plasmid protein comprising a secretion leader sequence.

Summary for Patent:   Start Trial

Foriegn Application Priority Data
Foreign Country Foreign Patent Number Foreign Patent Date
United Kingdom0329722.3Dec 23, 2003
PCT Information
PCT FiledDecember 23, 2004PCT Application Number:PCT/GB2004/005435
PCT Publication Date:July 07, 2005PCT Publication Number:WO2005/061719

Details for Patent 8,252,551

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Bayer Hlthcare TRASYLOL aprotinin INJECTABLE;INJECTION 020304 001 1994-02-07   Start Trial Novozymes Biopharma DK A/S (Bagsvaerd, DK) 2023-12-23 DISCN search
Microbix Biosystems KINLYTIC urokinase INJECTABLE;INJECTION 021846 001 1978-01-16   Start Trial Novozymes Biopharma DK A/S (Bagsvaerd, DK) 2023-12-23 DISCN search
Microbix Biosystems KINLYTIC urokinase INJECTABLE;INJECTION 021846 002 1978-01-16   Start Trial Novozymes Biopharma DK A/S (Bagsvaerd, DK) 2023-12-23 DISCN search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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