CELLULAR LIBRARIES OF PEPTIDE SEQUENCES (CLiPS) AND METHODS OF USING THE SAME

• US 20190032043A1
• Filed: 06/11/2018
• Published: 01/31/2019
• Est. Priority Date: 08/31/2005
• 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 Sean, Boulware, Kevin Todd
• Granted Patent: US 11,001,832 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/22 containing a Strep-tag
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

Abstract

The present invention provides compositions including peptide display scaffolds that present at least one candidate peptide and at least one detectable moiety in at least one of the N-terminal and C-terminal candidate peptide presenting domains that when expressed in a cell are accessible at a surface of the cell outermembrane. In addition, the present invention also provides kits and methods for screening a library of cells presenting the candidate peptides in peptide display scaffolds to identify a ligand for an enzyme.

Claims

1-46. -46. (canceled)

47. A method of identifying a substrate or ligand for an enzyme, comprising:

contacting a plurality of cells, wherein each cell expresses a peptide display scaffold comprising a fusion protein comprising the formula;

[D₁-C₁]-TM-[D₂],

wherein TM is a circularly permuted transmembrane protein;

wherein C₁ comprises a candidate peptide ligand or substrate;

wherein D₁ is a first detectable moiety and is a heterologous peptide or polypeptide;

wherein D₂ is a second detectable moiety and is a heterologous peptide or polypeptide;

wherein D₁ and D₂ are not the same; and

wherein D₁, D₂, and C₁ are exposed at an extracellular surface of the cell outer membrane; and

detecting the presence or absence of a D₁ signal and a D2 signal for at least one cell, wherein said detecting indicates whether C₁ comprises a substrate or ligand for the enzyme.

48. The method of claim 47, wherein the fusion protein comprises at least one linker, wherein the linker is between C₁ and TM or between D₂ and TM.

49. The method of claim 47, wherein detectable moieties D₁ and D₂ are affinity tags.

50. The method of claim 47, wherein the detecting step comprises detecting the presence of a detectable signal of D₁ and detecting the absence of a detectable signal of D₂ that is above a background level of a detectable D₂ signal.

51. The method of claim 47, wherein the C₁ comprises a member of a library of candidate peptide substrates or ligands for an enzyme.

52. The method of claim 47, wherein the fusion protein comprises a linker between the C₁ and the TM and a linker between the D₂ and the TM.

53. The method of claim 47, wherein at least one of detectable moieties D₁ and D₂ is an affinity tag.

54. The method of claim 47, wherein at least one of detectable moieties D₁ and D₂ is a binding peptide selected from the group consisting of a streptavidin binding peptide and a monocytic adaptor protein (MONA) binding peptide.

55. The method of claim 47, wherein at least one of detectable moieties D₁ and D₂ is a fluorescent protein.

56. The method of claim 47, wherein the D₁ and C₁ are located at an N-terminal domain of the fusion protein and the D₂ is located at a C-terminal domain of the fusion protein.

57. The method of claim 47, wherein the D₁ and C₁ are located at a C-terminal domain of the fusion protein and the D₂ is located at an N-terminal domain of the fusion protein.

58. The method of claim 47, wherein the circularly permuted transmembrane protein is a circularly permuted bacterial outer membrane protein.

59. The method of claim 58, wherein the circularly permuted bacterial outer membrane protein is selected from the group consisting of:

a circularly permuted OmpX, a circularly permuted OmpA, and a circularly permuted OmpT.

60. A method of identifying an allosteric regulator for an enzyme, comprising:

contacting a plurality of cells, wherein each cell expresses a peptide display scaffold comprising a fusion protein comprising the formula;

[D₁-C₁]-TM-[C₂-D₂],

wherein TM is a circularly permuted transmembrane protein;

wherein C₁ is a known substrate for the enzyme,

wherein C₂ comprises a candidate allosteric regulator;

wherein D₁ is a first detectable moiety and is a heterologous peptide or polypeptide;

wherein D₂ is a second detectable moiety and is a heterologous peptide or polypeptide;

wherein D₁ and D₂ are not the same; and

wherein D₁, D₂, C₁, and C₂, are exposed at an extracellular surface of the cell outer membrane; and

detecting the presence or absence of a D₁ signal and a D₂ signal for at least one cell, wherein said detecting indicates whether C₁ comprises an allosteric regulator for the enzyme.

61. The method of claim 60, wherein the C₂ comprises a member of a library of candidate allosteric substrates for the enzyme.

62. A method of identifying an inhibitor for an enzyme, comprising:

contacting a plurality of cells, wherein each cell expresses a peptide display scaffold comprising a fusion protein comprising the formula;

[D₁-C₁]-TM-[C₂-D₂],

wherein TM is a circularly permuted transmembrane protein;

wherein C₁ is a known substrate for the enzyme,

wherein C₂ comprises a candidate inhibitor;

wherein D₁ is a first detectable moiety and is a heterologous peptide or polypeptide;

wherein D₂ is a second detectable moiety and is a heterologous peptide or polypeptide;

wherein D₁ and D₂ are not the same; and

wherein D₁, D₂, C₁, and C₂, are exposed at an extracellular surface of the cell outer membrane; and

detecting the presence or absence of a D₁ signal and a D₂ signal for at least one cell, wherein said detecting indicates whether C₁ comprises an inhibitor for the enzyme.

63. The method of claim 62, wherein the C₂ comprises a member of a library of candidate inhibitors for the enzyme.