Mapping Protease Substrates by Using a Biotinylated Phage Substrate Library

Abstract

We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

Factor Xa subsite mapping by proteome-derived peptide libraries improved using WebPICS, a resource for proteomic identification of cleavage sites

“…Slight preferences for isoleucine and valine as well as arginine are observed in the chymotryptic PICS assay. Although these S3 preferences are corroborated by previously discussed factor Xa profiling experiments (Furlong et al, 2002;Scholle et al, 2006;Hsu et al, 2008) they are not confirmed by the Glu-C PICS assay. In S4, both PICS assays determine a minor preference for proline.…”

“…As well, this has been corroborated with synthetic peptide libraries (Cho et al, 1984;Kawabata et al, 1988). However, other studies indicated that negatively charged residues are disfavored in S3 (Bianchini et al, 2002) with P3 arginine being partially preferred in two substrate phage display screens (Scholle et al, 2006;Hsu et al, 2008) as well as in a synthetic peptide library screen (Furlong et al, 2002). The latter study also indicated a second, partial S3 preference for aromatic and aliphatic residues including phenylalanine, tyrosine, and leucine.…”

“…This specificity profile has been supported by biochemical and genetic profiling strategies. Thus, at S1 factor Xa is considered to be specific for arginine (Matthews and Wells, 1993;Scholle et al, 2006) although one study employing synthetic peptide libraries also revealed specificity profiles having a P1 lysine (Gosalia et al, 2005). S2 also has a strong contribution to factor Xa specificity.…”

“…S2 also has a strong contribution to factor Xa specificity. In addition to the canonical specificity for glycine (Cho et al, 1984;Kawabata et al, 1988;Matthews and Wells, 1993;Scholle et al, 2006), there is increasing evidence for a partial preference for aromatic and aliphatic residues in S2, in particular phenylalanine (Harris et al, 2000;Bianchini et al, 2002;Furlong et al, 2002;Gosalia et al, 2005;Hsu et al, 2008).…”

Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

“…Here we report an improved modular approach to immuno-detection based on PCR that employs intact antibodies, (rather than single-chain variable fragment antibodies (scFvs), which must be cloned and which usually have lower affinity than the parent antibody), avidin-linked to biotinylated bacteriophage as affinity agents. These SAM-AviTag phage are derivatives of the Escherichia coli phage M13 where the N-terminus of the phage tail protein III contains the enzymatically-biotinylatable AviTag peptide (GLNDIFEAQKIEWHE) (Scholle et al 2006). The lysine residue (K) in the AviTag is a substrate for biotinylation by the E. coli biotin ligase ( birA ) enzyme.…”