Protease-Resistant Systems for Polypeptide Display and Methods of Making and Using Thereof

A composition comprising:

• a replicable biological entity;
• and a displayed moiety (DM) displayed on the surface of the replicable biological entity, wherein the DM is fused to an extracellular terminus of a circularly permuted bacterial outer membrane carrier protein (CP) to produce a display platform (DP), wherein the CP comprises an amino acid sequence selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 56 and SEQ ID NO:
• 57.

A polypeptide display library comprising:

• a plurality of replicable biological entities, each member of the plurality comprising a displayed moiety (DM) displayed on the surface of the member replicable biological entity, wherein the DM is fused to an extracellular terminus of a circularly permuted bacterial outer membrane carrier protein (CP) to produce a display platform (DP), wherein the CP comprises an amino acid sequence selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 56 and SEQ ID NO:
• 57.

A method for identifying a peptide substrate for an enzyme, comprising:

• contacting a cell or other replicable biological entity with an enzyme, the cell comprising a cell outer membrane and expressing a peptide display scaffold, the peptide display scaffold comprising a fusion protein comprising a formula selected from the group consisting of:
• (1) T1-DM-carrier protein-T2 and (2) T1-carrier protein-DM-T2
• wherein the carrier protein (CP) is a circularly permuted bacterial outer membrane protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 56 and SEQ ID NO:
• 57;
• wherein DM comprises a member of a library of candidate peptide substrates for the enzyme;
• wherein T1 and T2 are first and second detectable moieties;
• and wherein T1-DM and T2 in formula (1) or T1 and DM-T2 in formula (2) 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;
• and wherein prior to contacting the cell with the enzyme, the cell exhibits a detectable T1 signal;
• and
• detecting the presence of a detectable T2 signal in the absence of a detectable T1 signal, identifies the member of the library of candidate peptide substrates as a peptide substrate for the enzyme.

A peptide display scaffold, comprising a fusion protein comprising a formula selected from the group consisting of:

• [T1-DM]-carrier protein-[T2] and (2) [T1]-carrier protein-[DM-1-T2] (1)
• wherein
• the carrier protein is a circularly permuted bacterial outer membrane protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 56 and SEQ ID NO:
• 57;
• DM comprises a candidate peptide;
• and
• T1 and T2 are first and second detectable moieties;
• and wherein, when the fusion protein is expressed in a cell comprising a cell outer membrane, T1-DM and T2 in formula (1) or T1 and DM-T2 in formula (2) are exposed at an extracellular surface of the cell outer membrane and one or both of the N- and C-termini of the fusion protein are exposed at the extracellular surface of the cell outer membrane.