Methods for making compositions including gram negative microbial polypeptides

1. A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising

providing a gram negative microbe;

disrupting the gram negative microbe in a buffer, wherein the gram negative microbe is present in the buffer at a concentration of between about 720 grams of microbe per 1,000 milliliters of buffer and about 1,080 grams of microbe per 1,000 milliliters of buffer;

solubilizing the disrupted gram negative microbe for greater than about 24 hours in a solution comprising sarcosine, wherein a ratio of the sarcosine to gram weight of disrupted gram negative microbe is between about 0.8 gram sarcosine per about 4.5 grams of disrupted gram negative microbe and about 1.2 grams sarcosine per about 4.5 grams of disrupted gram negative microbe; and

isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.

2. The method of claim 1 wherein the isolated molecules further comprise lipopolysaccarhide at a concentration of no greater than about 10.0 EU/ml.

3. A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising

providing a gram negative microbe;

disrupting the gram negative microbe;

solubilizing the disrupted gram negative microbe in a solution comprising sarcosine, wherein a ratio of the sarcosine to gram weight of disrupted gram negative microbe is between about 0.8 gram sarcosine per about 4.5 grams of disrupted gram negative microbe and about 1.2 grams sarcosine per about 4.5 grams of disrupted gram negative microbe; and

isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.

4. The method of claim 3 wherein the isolated molecules further comprise lipopolysaccarhide at a concentration of no greater than about 10.0 EU/ml.

5. A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising

providing a gram negative microbe;

disrupting the gram negative microbe;

solubilizing the disrupted gram negative microbe for greater than about 24 hours; and

isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.

6. The method of claim 5 wherein the isolated molecules further comprise lipopolysaccarhide at a concentration of no greater than about 10.0 EU/mi.

7. A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising

providing a gram negative microbe;

disrupting the gram negative microbe in a buffer, wherein the gram negative microbe is present in the buffer at a concentration of between about 720 grams of microbe per 1,000 milliliters of buffer and about 1,080 grams of microbe per 1,000 milliliters of buffer;

solubilizing the disrupted gram negative microbe; and

isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.

8. The method of claim 7 wherein the isolated molecules further comprise lipopolysaccarhide at a concentration of no greater than about 10.0 EU/ml.

9. A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising

providing a gram negative microbe;

disrupting the gram negative microbe;

solubilizing the disrupted gram negative microbe for greater than about 24 hours in a solution comprising sarcosine; and

isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.

10. A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising

providing a gram negative microbe;

disrupting the gram negative microbe in a buffer, wherein the gram negative microbe is present in the buffer at a concentration of between about 720 grams of microbe per 1,000 milliliters of buffer and about 1,080 grams of microbe per 1,000 milliliters of buffer;

solubilizing the disrupted gram negative microbe for greater than about 24 hours, wherein a ratio of the sarcosine to gram weight of disrupted gram negative microbe is between about 0.8 gram sarcosine per about 4.5 grams of disrupted gram negative microbe and about 1.2 grams sarcosine per about 4.5 grams of disrupted gram negative microbe; and

isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides, at least two porins and lipopolysaccarhide at a concentration of no greater than about 10.0 EU/ml.

11. A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising

providing a gram negative microbe;

disrupting the gram negative microbe;

solubilizing the disrupted gram negative microbe in a detergent for greater than about 24 hours; and

isolating molecules of the gram negative microbe by centrifugation, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.

12. The method of claim 11 wherein the isolated molecules further comprise lipopolysaccarhide at a concentration of no greater than about 10.0 EU/ml.

13. The method of claim 1 wherein the providing comprises incubating the gram negative microbe in media comprising an iron chelator.

14. The method of claim 13 wherein the iron chelator is 2,2′-dipyridyl.

15. The method of claim 1 wherein the gram negative microbe is Salmonella spp. or E. coli.

16. The method of claim 3 wherein the providing comprises incubating the gram negative microbe in media comprising an iron chelator.

17. The method of claim 16 wherein the iron chelator is 2,2′-dipyridyl.

18. The method of claim 3 wherein the gram negative microbe is Salmonella spp. or E. coli.

19. The method of claim 5 wherein the providing comprises incubating the gram negative microbe in media comprising an iron chelator.

20. The method of claim 19 wherein the iron chelator is 2,2′-dipyridyl.

21. The method of claim 5 wherein the gram negative microbe is Salmonella spp. or E. coli.

22. The method of claim 7 wherein the providing comprises incubating the gram negative microbe in media comprising an iron chelator.

23. The method of claim 22 wherein the iron chelator is 2,2′-dipyridyl.

24. The method of claim 7 wherein the gram negative microbe is Salmonella spp. or E. coli.

25. The method of claim 9 wherein the providing comprises incubating the gram negative microbe in media comprising an iron chelator.

26. The method of claim 25 wherein the iron chelator is 2,2′-dipyridyl.

27. The method of claim 9 wherein the gram negative microbe is Salmonella spp. or E. coli.

28. The method of claim 10 wherein the providing comprises incubating the gram negative microbe in media comprising an iron chelator.

29. The method of claim 28 wherein the iron chelator is 2,2′-dipyridyl.

30. The method of claim 10 wherein the gram negative microbe is Salmonella spp. or E. coli.

31. The method of claim 1 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.

32. The method of claim 31 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Kiebsiella spp., Serratia spp., or Yersinia spp.

33. The method of claim 31 wherein the member of the family Vibrionaceae is Vibrio cholerae.

34. The method of claim 31 wherein the member of the family Pasteurellaceae is Pasteurella spp.

35. The method of claim 34 wherein the Pasteurella spp. is Pasteurella multocida or Pasteurella haemolytica.

36. The method of claim 31 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.

37. The method of claim 36 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.

38. The method of claim 1 wherein the gram negative microbe is a Actinobacillus spp., Haemophilus spp., Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacter spp., Flavobacterium spp., or Aeromonas spp.

39. The method of claim 3 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.

40. The method of claim 39 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Kiebsiella spp., Serratia spp., or Yersinia spp.

41. The method of claim 39 wherein the member of the family Vibrionaceae is Vibrio cholerae.

42. The method of claim 39 wherein the member of the family Pasteurellaceae is Pasteurella spp.

43. The method of claim 42 wherein the Pasteurella spp. is Pasteurella multocida or Pasteurella haemolytica.

44. The method of claim 39 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.

45. The method of claim 44 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.

46. The method of claim 3 wherein the gram negative microbe is a Actinobacillus spp., Haemophilus spp., Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacter spp., Flavobacterium spp., or Aeromonas spp.

47. The method of claim 5 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.

48. The method of claim 47 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Kiebsiella spp., Serratia spp., or Yersinia spp.

49. The method of claim 47 wherein the member of the family Vibrionaceae is Vibrio cholerae.

50. The method of claim 47 wherein the member of the family Pasteurellaceae is Pasteurella spp.

51. The method of claim 50 wherein the Pasteurella spp. is Pasteurella multocida or Pasteurella haemolytica.

52. The method of claim 47 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.

53. The method of claim 52 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.

54. The method of claim 5 wherein the gram negative microbe is a Actinobacillus spp., Haemophilus spp., Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacter spp., Flavobacterium spp., or Aeromonas spp.

55. The method of claim 7 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.

56. The method of claim 55 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Kiebsiella spp., Serratia spp., or Yersinia spp.

57. The method of claim 55 wherein the member of the family Vibrionaceae is Vibric cholerae.

58. The method of claim 55 wherein the member of the family Pasteurellaceae is Pasteurella spp.

59. The method of claim 58 wherein the Pasteurella spp. is Pasteurella multocida or Pasteurella haemolytica.

60. The method of claim 55 wherein the member of the family Pseudomonadaceac is Pseudomonas spp.

61. The method of claim 60 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.

62. The method of claim 7 wherein the gram negative microbe is a Actinobacillus spp., Haemophilus spp., Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacter spp., Flavobacterium spp., or Aeromonas spp.

63. The method of claim 9 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.

64. The method of claim 63 wherein the member of the family Enterobacteriaceac is Shigella spp., Proteus spp., Kiebsiella spp., Serratia spp., or Yersinia spp.

65. The method of claim 63 wherein the member of the family Vibrionaceae is Vibrio cholerae.

66. The method of claim 63 wherein the member of the family Pasteurellaceae is Pasteurella spp.

67. The method of claim 66 wherein the Pasteurella spp. is Pasteurella multocida or Pasteurella haemolytica.

68. The method of claim 63 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.

69. The method of claim 68 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.

70. The method of claim 9 wherein the gram negative microbe is a Actinobacillus spp., Haemoplzilus spp., Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacter spp., Flavobacterium spp., or Aeromonas spp.

71. The method of claim 10 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.

72. The method of claim 71 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Kiebsiella spp., Serratia spp., or Yersinia spp.

73. The method of claim 71 wherein the member of the family Vibrionaceae is Vibric cholerae.

74. The method of claim 71 wherein the member of the family Pasteurellaceae is Pasteurella spp.

75. The method of claim 74 wherein the Pasteurella spp. is Pasteurella multocida or Pasteurella haemolytica.

76. The method of claim 71 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.

77. The method of claim 76 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.

78. The method of claim 10 wherein the gram negative microbe is a Actinobacillus spp., Haemophilus spp., Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacter spp., Flavobacterium spp., or Aeromonas spp.

79. The method of claim 11 wherein the gram negative microbe is a member of the family Enterobacteriaceae, family Vibrionaceae, family Pasteurellaceae, or family Pseudomonadaceae.

80. The method of claim 79 wherein the member of the family Enterobacteriaceae is Shigella spp., Proteus spp., Kiebsiella spp., Serratia spp., or Yersinia spp.

81. The method of claim 79 wherein the member of the family Vibrionaceae is Vibrio cholerae.

82. The method of claim 79 wherein the member of the family Pasteurellaceae is Pasteurella spp.

83. The method of claim 82 wherein the Pasteurella spp. is Pasteurella multocida or Pasteurella haemolytica.

84. The method of claim 79 wherein the member of the family Pseudomonadaceae is Pseudomonas spp.

85. The method of claim 84 wherein the Pseudomonas spp. is Pseudomonas aeruginosa.

86. The method of claim 11 wherein the gram negative microbe is a Actinobacillus spp., Haemophilus spp., Myxcobacteria spp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacter spp., Flavobacterium spp., or Aeromonas spp.