Expression of lipoproteins 机翻标题: 暂无翻译,请尝试点击翻译按钮。

公开号/公开日
EP1741718 A2 2007-01-10 [EP1741718]EP1741718 A3 2007-03-28 [EP1741718] / 2007-01-102007-03-28
申请号/申请日
2006EP-0016140 / 1996-06-05
发明人
HUEBNER ROBERT C;ERDILE LORNE F;WARAKOMSKI DONALD J;BECKER ROBERT S;GRAY MARYANN B;PYLE DEREK L;
申请人
SANOFI PASTEUR;
主分类号
IPC分类号
A01N-063/00A61K-031/00A61K-038/00A61K-038/17A61K-039/02A61K-039/09A61K-039/39A61P-037/00A61P-037/04C07H-021/04C07K-014/195C07K-014/205C07K-014/315C07K-019/00C12N-015/09C12N-015/62C12P-021/00C12P-021/02C12P-021/04C12R-001/19
摘要
(EP1741718) Heterologous lipidated proteins formed recombinantly are disclosed and claimed.  The expression system can be E.coli.  The heterologous lipidated protein has a leader sequence which does not naturally occur with the protein portion of the lipidated protein.  The lipidated protein can have the Borrelia OspA leader sequence.  The protein portion can be OspC, PspA, UreA, UreB, or a fragment thereof.  Methods and compositions for forming and employing the proteins are also disclosed and claimed.
机翻摘要
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地址
代理人
代理机构
;
优先权号
1995US-08475781 1995-06-07 1995US-08486373 1995-06-07 1996EP-0918793 1996-06-05
主权利要求
(EP1741718)  1. A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a signal sequence of a lipoprotein and a second nucleic acid sequence encoding a mature protein, or fragment thereof, which is heterologous to the lipoprotein encoded by said first nucleic acid sequence, said first nucleic acid sequence being contiguous with said second nucleic acid sequence when the mature protein is naturally lipidated, or said first and second nucleic acid sequences being separated by one codon coding for one amino acid when the mature protein is not naturally lipidated. 2. The hybrid nucleic acid molecule of claim 1 wherein said signal sequence is the signal sequence of an OspA protein of a Borrelia species. 3. The hybrid nucleic acid molecule of claim 2 wherein said first nucleic acid sequence and said second nucleic acid sequence are coupled in a translational open reading frame relationship. 4. The hybrid nucleic acid molecule of claim 3 wherein said mature protein is an OspC lipoprotein of a Borrelia species, or a fragment thereof. 5. The hybrid molecule of claim 4 wherein said OspC lipoprotein is that of a strain of B. burgdorferi. 6. The hybrid molecule of claim 5 wherein said strain of B. burgdorferi is selected from the OspC sub-type families. 7. The hybrid molecule of claim 5 wherein said OspA protein is that of a strain of B. burgdorferi. 8. The hybrid molecule of claim 7 wherein said strain of B. burgdorferi is selected from the B31, ACA1 and Ip90 families of strains. 9. The hybrid nucleic acid molecule of claim 3 wherein said mature protein is a PspA protein of a strain of S. pneumoniae, or a fragment thereof. 10. The hybrid molecule of claim 9 wherein said OspA protein is that of a strain of B. burgdorferi. 11. The hybrid molecule of claim 10 wherein said strain of B. burgdorferi is selected from the B31, ACA1 and Ip90 families of strains. 12. The hybrid nucleic acid molecule of claim 3 wherein said mature protein is a UreA protein of a strain of H. pylori, or a fragment thereof. 13. The hybrid molecule of claim 12 wherein said OspA protein is that of a strain of B. burgdorferi. 14. The hybrid molecule of claim 13 wherein said strain of B. burgdorferi is selected from the B31, ACA1 and Ip90 families of strains. 15. The hybrid nucleic acid molecule of claim 3 wherein said mature protein is a UreB protein of a strain of H. pylori, or a fragment thereof. 16. The hybrid molecule of claim 15 wherein said OspA protein is that of a strain of B. burgdorferi. 17. The hybrid molecule of claim 16 wherein said strain of B. burgdorferi is selected from the B31, ACA1 and Ip90 families of strains. 18. A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding an OspC lipoprotein of a Borrelia species and a second nucleic acid sequence encoding a signal sequence of an expressed protein heterologous to OspC and coupled in translational open reading frame relationship with said first nucleic acid sequence. 19. The hybrid nucleic acid molecule of claim 18 wherein said OspC lipoprotein is that of a strain of B. burgdorferi. 20. The hybrid nucleic acid molecule of claim 19 wherein said strain of B. burgdorferi is selected from the OspC sub-type families. 21. A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a PspA protein of a strain of S. pneumoniae and a second nucleic acid sequence encoding a signal sequence of an expressed protein heterlogous to PspA and coupled in translational open reading frame relationship with said first nucleic acid sequence. 22. A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a UreA protein of a strain of H. pylori and a second nucleic acid sequence encoding a signal sequence of an expressed protein heterlogous to UreA and coupled in translational open reading frame relationship with said first nucleic acid sequence. 23. A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a UreB protein of a strain of H. pylori and a second nucleic acid sequence encoding a signal sequence of an expressed protein heterlogous to UreB and coupled in translational open reading frame relationship with said first nucleic acid sequence. 24. An expression vector containing the hybrid nucleic acid molecule of claim 1 under control of a promoter for expression of said mature protein. 25. The expression vector of claim 24 wherein said mature protein is an OspC lipoprotein of a Borrelia species. 26. The expression vector of claim 24 wherein said mature protein is a PspA lipoprotein of a strain of S. pneumoniae. 27. The expression vector of claim 24 wherein said mature protein is a UreA protein of a strain of H. pylori. 28. The expression vector of claim 24 wherein said mature protein is a UreB protein of a strain of H. pylori. 29. An expression vector containing the hybrid nucleic acid molecule of claim 18 under control of a promoter for expression of said OspC lipoprotein. 30. An expression vector containing the hybrid nucleic acid molecule of claim 21 under control of a promoter for expression of said PspA protein. 31. An expression vector containing the hybrid nucleic acid molecule of claim 22 under control of a promoter for expression of said UreA protein. 32. An expression vector containing the hybrid nucleic acid molecule of claim 23 under control of a promoter for expression of said UreB protein. 33. A method for forming a recombinant protein, which comprises: incorporating the expression vector of claim 24 into a host organism;  and effecting expression of said mature protein from the host organism. 34. The method of claim 33 wherein said mature protein is an OspC lipoprotein of a Borrelia species. 35. The method of claim 34 wherein said host organism is E. coli. 36. The method of claim 33 wherein said mature protein is a PspA protein of a strain of S. pneumoniae. 37. The method of claim 36 wherein said host organism is E. coli. 38. The method of claim 33 wherein said-mature protein is a UreA protein of a strain of H. pylori. 39. The method of claim 38 wherein said host organism is E. coli. 40. The method of claim 33 wherein said mature protein is a UreB protein of a strain of H. pylori. 41. The method of claim 40 wherein said host organism is E. coli. 42. A method for forming recombinant OspC lipoprotein, which comprises: incorporating the expression vector of claim 29 into a host organism;  and effecting expression of said OspC lipoprotein from the host organism. 43. The method of claim 42 wherein said host organism is E. coli. 44. A method for forming recombinant PspA lipoprotein, which comprises: incorporating the expression vector of claim 30 into a host organism;  and effecting expression of said PspA lipoprotein from the host organism. 45. The method of claim 44 wherein said host organism is E. coli. 46. A method for forming recombinant UreA lipoprotein, which comprises: incorporating the expression vector of claim 31 into a host organism;  and effecting expression of said UreA lipoprotein from the host organism. 47. The method of claim 46 wherein said host organism is E. coli. 48. A method for forming recombinant UreB lipoprotein, which comprises: incorporating the expression vector of claim 32 into a host organism;  and effecting expression of said UreA lipoprotein from the host organism. 49. The method of claim 48 wherein said host organism is E. coli. 50. A process for the production of a recombinant lipoprotein, which comprises: constructing a hybrid nucleic acid molecule comprising a first nucleic acid sequence encoding a signal sequence of a lipoprotein and a second nucleic acid sequence encoding a mature protein, or fragment thereof, which is heterologous to the lipoprotein encoded by said first nucleic acid, said second nucleic acid sequence being contiguous with said first sequence;  forming an expression vector containing said hybrid nucleic acid molecule under control of a promoter for expression of said mature protein;  incorporating said expression vector into a host organism;  effecting expression of said recombinant lipoprotein by said host organism;  lysing the cells of the host organism;  treating the lysed cells with a surfactant which selectively solubilizes said recombinant lipoprotein in preference to bacterial and other proteins and which is able to effect phase separation of a detergent phase under mild conditions;  effecting phase separation at a detergent phase containing solubilized recombinant lipoprotein, an aqueous phase containing bacterial and other proteins and a solid phase containing cell residue;  separating and recovering said detergent phase from said solid phase and said aqueous phase;  contacting said detergent phase with a first chromatographic column under conditions which result in binding of protein other than said recombinant lipoprotein to said column to provide a flow-through from said first chromatographic column containing the recombinant lipoprotein and recovering said flow-through from said first chromatographic column;  contacting the flow-through from said first chromatographic column with a second chromatographic column under conditions which result in binding of the recombinant lipoprotein to the second chromatographic column in preference to contaminant proteins and lipopolysaccharides which pass through said second chromatographic column;  eluting said recombinant lipoprotein from said second chromatographic column to provide an eluant containing said recombinant lipoprotein substantially free from lipopolysaccharide and contaminant proteins;  and recovering said eluant. 51. The process of claim 50 wherein said signal sequence is the signal sequence of an OspA protein of a Borrelia species. 52. The process of claim 51 wherein said first nucleic acid sequence and said second nucleic acid sequence are coupled in a translational open reading frame relationship. 53. The process of claim 52 wherein said surfactant is TRITON (Registered Trademark X-114. 54. The process of claim 53 wherein said treating of lysed cells is effected at a temperature of about 0 deg.C to about 10 deg.C, the resulting mixture is treated to a mildly elevated temperature of about 35 deg.C to about 40 deg.C to effect separation of said detergent phase, and said detergent phase is separated from said aqueous phase and said solid phase by centrifugation. 55. The process of claim 52 wherein said mature protein is an OspC lipoprotein of a Borrelia species. 56. The process of claim 55 wherein said first chromatographic column is further contacted with a buffer medium at a pH to provide liquid containing the recombinant OspC lipoprotein from the first chromatographic column while the other proteins are retained on the first chromatographic column and the flow-through from the further contact is collected and combined with that from the first contacting step on said first chromatographic column and the combined flow-through is contacted with said second chromatographic column. 57. The process of claim 52 wherein said mature protein is a PspA protein of a strain of S. pneumoniae. 58. The process of claim 57, wherein said first chromatographic column is further contacted with a buffer medium at a pH to provide liquid containing the recombinant PspA lipoprotein from the first chromatographic column while the other proteins are retained on the first chromatographic column and the flow-through from the further contact is collected and combined with that from the first contacting step on said first chromatographic column and the combined flow-through is contacted with said second chromatographic column. 59. The process of claim 52 wherein said mature protein is a UreA protein of a strain of H. pylori. 60. The process of claim 59 wherein said first chromatographic column is further contacted with a buffer medium at a pH to provide liquid containing the recombinant UreA lipoprotein from the first chromatographic column while the other proteins are retained on the first chromatographic column and the flow-through from the further contact is collected and combined with that from the first contacting step on said first chromatographic column and the combined flow-through is contacted with said second chromatographic column. 61. The process of claim 52 wherein said mature protein is a UreB protein of a strain of H. pylori. 62. The process of claim 61 wherein said first chromatographic column is further contacted with a buffer medium at a pH to provide liquid containing the recombinant UreB lipoprotein from the first chromatographic column while the other proteins are retained on the first chromatographic column and the flow-through from the further contact is collected and combined with that from the first contacting step on said first chromatographic column and the combined flow-through is contacted with said second chromatographic column. 63. The process of claim 52 wherein said host organism lysis is effected by freezing and thawing the host organism. 64. Recombinantly-produced, isolated and purified lipoprotein produced by the process of claim 50. 65. Recombinantly-produced, isolated and purified OspC lipoprotein of a Borrelia strain having a purity of at least about 80% and substantially free from contaminant proteins and lipopolysaccharides. 66. Recombinantly-produced, isolated and purified lipidated PspA protein of a strain of S. pneumoniae having a purity of at least about 50% and substantially free from contaminant proteins and lipopolysaccharides. 67. Recombinantly-produced, isolated and purified lipidated UreA protein of a strain of H. pylori having a purity of at least about 80% and substantially free from contaminant proteins and lipopolysaccharides. 68. Recombinantly-produced, isolated and purified lipidated UreB protein of a strain of H. pylori having a purity of at least about 80% and substantially free from contaminant proteins and lipopolysaccharides. 69. A lipoprotein fusion vector PLF100 having ATCC Accession No. 69750. 70. A method for inducing an immunological response in a human or animal comprising the step of administering to said human or animal a composition comprising the lipoprotein of claim 64 or 65. 71. A composition for inducing an immunological response comprising the lipoprotein of claim 64 or 65. 72. A method for inducing an immunological response in a human or animal comprising the step of administering to said human or animal a composition comprising the lipidated PspA protein of claim 64 or 66. 73. A composition for inducing an immunological response comprising the lipidated PspA protein of claim 64 or 66. 74. A method for inducing an immunological response in a human or animal comprising the step of administering to said human or animal a composition comprising the lipidated UreA protein of claim 67. 75. A composition for inducing an immunological response comprising the lipidated UreA protein of claim 67. 76. A method for inducing an immunological response in a human or animal comprising the step of administering to said human or animal a composition comprising the lipidated UreB protein of claim 68. 77. A composition for inducing an immunological response comprising the lipidated UreB protein of claim 68. 78. A method for enhancing the immunogenicity of a protein, comprising the steps of: forming a hybrid nucleic acid molecule comprising a first nucleic acid sequence encoding a signal sequence of a lipoprotein and a second nucleic acid sequence encoding a mature protein, or fragment thereof, which is heterologous to the lipoprotein encoded by said first nucleic acid, said first nucleic acid sequence being contiguous with said second nucleic acid sequence;  incorporating said hybrid nucleic acid molecule into an expression vector;  incorporating said expression vector into a host organism;  and effecting expression of said mature protein from said host organism.
法律状态
(EP1741718) LEGAL DETAILS FOR EP1741718  Actual or expected expiration date=2010-07-24    Legal state=DEAD    Status=LAPSED     Event publication date=1996-06-05  Event code=EP/APP  Event indicator=Pos  Event type=Examination events  Application details  Application country=EP EP06016140  Application date=1996-06-05  Standardized application number=2006EP-0016140     Event publication date=2007-01-10  Event code=EP/A2  Event type=Examination events  Application published without search report  Publication country=EP  Publication number=EP1741718  Publication stage Code=A2  Publication date=2007-01-10  Standardized publication number=EP1741718     Event publication date=2007-01-10  Event code=EP/AK  Event indicator=Pos  Event type=Designated states  Designated contracting states: Benannte vertragsstaaten AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE    Event publication date=2007-01-10  Event code=EP/17P  Event indicator=Pos  Event type=Examination events  Request for examination filed Pruefungsantrag gestellt  Effective date of the event=2006-08-24     Event publication date=2007-03-28  Event code=EP/A3  Event indicator=Pos  Event type=Examination events  Published search report  Publication country=EP  Publication number=EP1741718  Publication stage Code=A3  Publication date=2007-03-28  Standardized publication number=EP1741718     Event publication date=2007-03-28  Event code=EP/AK  Event indicator=Pos  Event type=Designated states  Designated contracting states: Benannte vertragsstaaten AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE    Event publication date=2007-07-04  Event code=EP/17Q  Event indicator=Pos  Event type=Examination events  First examination report Erster pruefungsbescheid  Effective date of the event=2007-06-01     Event publication date=2007-12-05  Event code=EP/AKX  Event indicator=Pos  Event type=Designated states  Event type=Payment or non-payment notifications  Event type=Examination events  Payment of designation fees Zahlung von benennungsgebuehren AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE    Event publication date=2011-01-26  Event code=EP/18D  Event indicator=Neg  Event type=Event indicating Not In Force  Application deemed to be withdrawn  Effective date of the event=2010-07-24  MEMBER STATE LEGAL DETAILS FOR EP0832093  Actual or expected expiration date=2016-06-05    Legal state=DEAD    Status=EXPIRED   Corresponding cc:  Designated or member state=EP Corresponding appl: EP96918793  Application date in the designated or member state=1996-06-05   Application number in the designated or member state=1996EP-0918793 Corresponding cc:  Designated or member state=EP Corresponding pat: EP0832093  Publication stage code in the designated or member state=A1  Publication date in the designated or member state=1998-04-01   Publication number in the designated or member state=EP-832093    Event publication date=2007-01-10  Event code=EP/AC  Event type=Examination events  Divisional application (art. 76) of: Corresponding cc:  Designated or member state=EP
专利类型码
A2A3
国别省市代码
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