Methods for enhancing bacterial cell display of proteins and peptides

• US 8,293,685 B2
• Filed: 07/24/2008
• Issued: 10/23/2012
• Est. Priority Date: 07/26/2007
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Daugherty, Patrick S., Rice, Jeffrey
• Primary Examiner(s): Steele, Amber D
• Application Number:
  US12/220,448
• Publication Number:
  US 20090062142A1
• Time in Patent Office: 1,552 Days
• Field of Search: 506/2, 506/9, 506/14, 506/26, 435/6.1, 435/7.2, 435/69.1, 435/71.1, 530/350, 530/402, 536/23.4
• US Class Current: 506/14
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display

Abstract

Methods of making and using bacterial display polypeptide libraries using circularly permuted OmpX (CPX) variants are disclosed. The invention further relates to methods for enhancing the display of proteins and peptides at the surface of bacteria by optimizing linkers and incorporating mutations at positions 165 and 166 of CPX.

Claims

1. A circularly permuted OmpX (CPX) variant comprising a linker joining the native N-terminus and native C-terminus or OmpX, wherein the CPX variant comprises a non-native N-terminus and a non-native C-terminus, wherein the linker is 5-8 residues in length and the linker comprises the sequence of SEQ ID NOs:

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

2. The CPX variant of claim 1, wherein the linker is 5 residues in length.

3. The CPX variant of claim 1, wherein the linker is 6 residues in length.

4. The CPX variant of claim 1, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

5. The CPX variant of claim 4, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

6. The CPX variant of claim 1, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

7. The CPX variant of claim 6, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

8. The CPX variant of claim 1, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

9. The CPX variant of claim 8, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

10. The CPX variant of claim 1, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

11. The CPX variant of claim 10, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

12. The CPX variant of claim 11, wherein the detectable label comprises a streptavidin binding peptide.

13. The CPX variant of claim 11, wherein the second passenger polypeptide comprises the detectable label.

14. The CPX variant of claim 12, wherein the streptavidin binding peptide comprises the sequence of SEQ ID NO:

36.

15. The CPX variant of claim 11, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

16. The CPX variant of claim 15, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

17. The CPX variant of claim 10, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

18. The CPX variant of claim 17, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

19. A polypeptide display library comprising a polypeptide displayed by the CPX variant of claim 1.

20. A method of making a polypeptide display library, the method comprising:

providing a plurality of expression vectors expressing CPX variants of claim 1 carrying a plurality of passenger polypeptides,

transfecting bacterial cells with said expression vectors, and

culturing the bacterial cells under conditions that permit expression of said passenger polypeptides on the surface of the bacterial cells.

21. A method of screening for a CPX variant that displays a passenger polypeptide with greater efficiency than another carrier protein carrying the same passenger polypeptide, the method comprising:

transfecting a bacterial cell with an expression vector expressing a CPX variant of claim 1 carrying the passenger polypeptide,

screening for display of the passenger polypeptide at the surface of the bacterial cell within 25 minutes after inducing the expression of the CPX variant carrying the passenger polypeptide; and

comparing the display efficiency of the CPX variant carrying the passenger polypeptide to the display efficiency of another carrier protein carrying the same passenger polypeptide expressed under the same conditions.

22. A method of enhancing the display efficiency of a passenger polypeptide, the method comprising:

screening a plurality of different CPX variants of claim 1 and

selecting the CPX variant that displays the passenger polypeptide with the greatest efficiency compared to the plurality of other CPX variants.

23. The method of claim 22, wherein the linker is 6 residues in length.

24. A method of screening a library of polypeptides for the ability to bind to a target molecule, the method comprising:

a) providing a polypeptide display library comprising CPX variants as set forth in claim 1 carrying a plurality of passenger polypeptides displayed on bacterial cells,

b) contacting the plurality of passenger polypeptides with the target molecule, and

c) identifying at least one displayed passenger polypeptide that binds to the target molecule.

25. The method of claim 24, wherein the target molecule is selected from the group consisting of a receptor, a ligand, an antibody, an antigen, an enzyme, a transporter, a substrate, an inhibitor, an activator, a cofactor, a drug, a nucleic acid, a lipid, a carbohydrate, a glycoprotein, a small organic molecule, and an inorganic molecule.

26. The method of claim 24, wherein said target molecule comprises a detectable label, wherein identifying the target molecule bound to at least one passenger polypeptide comprises detecting the label attached to said target molecule.

27. A method of screening a library of polypeptides for biological activity in the presence of a target molecule, the method comprising:

a) providing a polypeptide display library comprising CPX variants as set forth in claim 1 carrying a plurality of passenger polypeptides displayed on bacterial cells,

b) contacting the plurality of passenger polypeptides with the target molecule,

c) assaying for biological activity in the presence of the target molecule, and

d) identifying at least one displayed passenger polypeptide that has biological activity.

28. The CPX variant of claim 7, wherein said linker between the non-native N-terminus of the CPX variant and the passenger polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

29. The CPX variant of claim 9, wherein said linker between the non-native C-terminus of the CPX variant and the passenger polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

30. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

18.

31. The CPX variant of claim 30, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

32. The CPX variant of claim 31, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

33. The CPX variant of claim 30, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

34. The CPX variant of claim 33, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

35. The CPX variant of claim 30, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

36. The CPX variant of claim 35, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

37. The CPX variant of claim 30, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

38. The CPX variant of claim 37, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

39. The CPX variant of claim 38, wherein the second passenger polypeptide comprises the detectable label.

40. The CPX variant of claim 38, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

41. The CPX variant of claim 40, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

42. The CPX variant of claim 37, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

43. The CPX variant of claim 42, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

44. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

13.

45. The CPX variant of claim 44, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

46. The CPX variant of claim 45, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

47. The CPX variant of claim 44, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

48. The CPX variant of claim 47, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

49. The CPX variant of claim 44, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

50. The CPX variant of claim 49, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

51. The CPX variant of claim 44, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

52. The CPX variant of claim 51, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

53. The CPX variant of claim 52, wherein the second passenger polypeptide comprises the detectable label.

54. The CPX variant of claim 52, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

55. The CPX variant of claim 54, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

56. The CPX variant of claim 51, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

57. The CPX variant of claim 56, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

58. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

17.

59. The CPX variant of claim 58, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

60. The CPX variant of claim 59, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

61. The CPX variant of claim 58, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

62. The CPX variant of claim 61, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

63. The CPX variant of claim 58, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

64. The CPX variant of claim 63, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

65. The CPX variant of claim 58, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

66. The CPX variant of claim 65, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

67. The CPX variant of claim 66, wherein the second passenger polypeptide comprises the detectable label.

68. The CPX variant of claim 66, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

69. The CPX variant of claim 68, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

70. The CPX variant of claim 69, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

71. The CPX variant of claim 70, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

72. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

20.

73. The CPX variant of claim 72, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

74. The CPX variant of claim 73, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

75. The CPX variant of claim 72, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

76. The CPX variant of claim 75, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

77. The CPX variant of claim 72, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

78. The CPX variant of claim 77, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

79. The CPX variant of claim 72, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

80. The CPX variant of claim 79, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

81. The CPX variant of claim 80, wherein the second passenger polypeptide comprises the detectable label.

82. The CPX variant of claim 80, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

83. The CPX variant of claim 82, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

84. The CPX variant of claim 79, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

85. The CPX variant of claim 84, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

86. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

21.

87. The CPX variant of claim 86, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

88. The CPX variant of claim 87, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

89. The CPX variant of claim 86, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

90. The CPX variant of claim 89, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

91. The CPX variant of claim 86, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

92. The CPX variant of claim 91, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

93. The CPX variant of claim 86, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

94. The CPX variant of claim 93, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

95. The CPX variant of claim 94, wherein the second passenger polypeptide comprises the detectable label.

96. The CPX variant of claim 94, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

97. The CPX variant of claim 96, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

98. The CPX variant of claim 93, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

99. The CPX variant of claim 98, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

100. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

22.

101. The CPX variant of claim 100, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

102. The CPX variant of claim 101, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

103. The CPX variant of claim 100, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

104. The CPX variant of claim 103, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

105. The CPX variant of claim 100, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

106. The CPX variant of claim 105, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

107. The CPX variant of claim 100, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

108. The CPX variant of claim 107, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

109. The CPX variant of claim 108, wherein the second passenger polypeptide comprises the detectable label.

110. The CPX variant of claim 108, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

111. The CPX variant of claim 110, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

112. The CPX variant of claim 107, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

113. The CPX variant of claim 112, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

114. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

23.

115. The CPX variant of claim 114, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

116. The CPX variant of claim 115, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

117. The CPX variant of claim 114, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

118. The CPX variant of claim 117, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

119. The CPX variant of claim 114, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

120. The CPX variant of claim 119, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

121. The CPX variant of claim 114, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

122. The CPX variant of claim 121, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

123. The CPX variant of claim 122, wherein the second passenger polypeptide comprises the detectable label.

124. The CPX variant of claim 122, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

125. The CPX variant of claim 124, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

126. The CPX variant of claim 121, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

127. The CPX variant of claim 126, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

128. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

24.

129. The CPX variant of claim 128, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

130. The CPX variant of claim 129, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

131. The CPX variant of claim 128, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

132. The CPX variant of claim 131, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

133. The CPX variant of claim 128, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

134. The CPX variant of claim 133, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

135. The CPX variant of claim 128, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

136. The CPX variant of claim 135, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

137. The CPX variant of claim 136, wherein the second passenger polypeptide comprises the detectable label.

138. The CPX variant of claim 136, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

139. The CPX variant of claim 138, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

140. The CPX variant of claim 135, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

141. The CPX variant of claim 140, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

142. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

26.

143. The CPX variant of claim 142, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

144. The CPX variant of claim 143, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

145. The CPX variant of claim 142, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

146. The CPX variant of claim 145, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

147. The CPX variant of claim 142, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

148. The CPX variant of claim 147, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

149. The CPX variant of claim 142, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

150. The CPX variant of claim 149, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

151. The CPX variant of claim 150, wherein the second passenger polypeptide comprises the detectable label.

152. The CPX variant of claim 150, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

153. The CPX variant of claim 152, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

154. The CPX variant of claim 149, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

155. The CPX variant of claim 154, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

156. The CPX variant of claim 1, wherein the linker joining the native N-terminus and native C-terminus of OmpX comprises the sequence set forth in SEQ ID NO:

27.

157. The CPX variant of claim 156, comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is a substitution mutation at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

158. The CPX variant of claim 157, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A165I mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

159. The CPX variant of claim 156, comprising a passenger polypeptide fused to the non-native N-terminus of the CPX variant.

160. The CPX variant of claim 159, comprising a linker between the non-native N-terminus of the CPX variant and the passenger polypeptide.

161. The CPX variant of claim 156, comprising a passenger polypeptide fused to the non-native C-terminus of the CPX variant, wherein the passenger polypeptide comprises a detectable label.

162. The CPX variant of claim 161, comprising a linker between the non-native C-terminus of the CPX variant and the passenger polypeptide.

163. The CPX variant of claim 156, comprising a first passenger polypeptide fused to the non-native N-terminus of the CPX variant and a second passenger polypeptide fused to the non-native C-terminus of the CPX variant.

164. The CPX variant of claim 163, wherein the first passenger polypeptide or the second passenger polypeptide comprises a detectable label.

165. The CPX variant of claim 164, wherein the second passenger polypeptide comprises the detectable label.

166. The CPX variant of claim 165, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

167. The CPX variant of claim 166, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

168. The CPX variant of claim 161, comprising a linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or the second passenger polypeptide and the non-native C-terminus of the CPX variant.

169. The CPX variant of claim 168, wherein the linker between the first passenger polypeptide and the non-native N-terminus of the CPX variant or between the second passenger polypeptide and the non-native C-terminus of the CPX variant comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.