Cellular Libraries of Peptide Sequences (CLiPS) and Methods of Using the Same
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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.
56 Citations
62 Claims
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1-46. -46. (canceled)
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47. A peptide display scaffold, comprising a fusion protein comprising the formula:
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[D1-C1]-TM-[C2-D2]wherein TM is a circularly permuted bacterial outer membrane protein; C1 comprises a known substrate for an enzyme; C2 comprises a candidate allosteric regulator of the enzyme; and D1 and D2 are first and second detectable moieties, wherein D1 and D2 are not the same; and wherein when the fusion protein is expressed in a cell comprising a cell outer membrane, D1-C1 and C2-D2 are accessible at the extracellular surface of the cell outer membrane and the N- or C-terminus of the fusion protein is accessible at the extracellular surface of the cell outer membrane. - View Dependent Claims (48, 49, 50, 60)
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51. A peptide display scaffold, comprising a fusion protein comprising the formula:
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[D1-C1]-TM-[C2-D2]wherein TM is a circularly permuted bacterial outer membrane protein; C1 comprises a known allosteric regulator of an enzyme; C2 comprises a candidate substrate for the enzyme; and D1 and D2 are first and second detectable moieties, wherein D1 and D2 are not the same; and wherein when the fusion protein is expressed in a cell comprising a cell outer membrane, D1-C1 and C2-D2 are accessible at the extracellular surface of the cell outer membrane and the N- or C-terminus of the fusion protein is accessible at the extracellular surface of the cell outer membrane. - View Dependent Claims (52, 53, 61)
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54. A peptide display scaffold, comprising a fusion protein comprising the formula:
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[D1-C1]-TM-[D2]wherein TM is a circularly permuted bacterial outer membrane protein; C1 comprises a candidate peptide; and D1 and D2 are first and second detectable moieties, wherein D1 and D2 are not the same and wherein when D1 provides a detectable D1 signal D2 does not provide a detectable D2 signal above a background level of a detectable D2 signal; and wherein when the fusion protein is expressed in a cell comprising a cell outer membrane, D1-C1 and D2 are accessible at the extracellular surface of the cell outer membrane and the N- or C-terminus of the fusion protein is accessible at the extracellular surface of the cell outer membrane. - View Dependent Claims (55, 56, 57, 58, 59, 62)
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Specification