Mapping protease substrates by using a biotinylated phage substrate library.

Michael D Scholle; Ushma Kriplani; Amanda Pabon; Kamakshi Sishtla; Marc J Glucksman; Brian K Kay

doi:10.1002/cbic.200500427

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Mapping protease substrates by using a biotinylated phage substrate library.

Michael D Scholle·Ushma Kriplani·Amanda Pabon·Kamakshi Sishtla·Marc J Glucksman·Brian K Kay

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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.

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Bacteriophage M13 Biotinylation Factor Xa Humans Mass Spectrometry Metalloendopeptidases Peptide Hydrolases Peptide Library Peptides Sensitivity and Specificity Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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Michael D Scholle, Ushma Kriplani, Amanda Pabon, Kamakshi Sishtla, Marc J Glucksman, Brian K Kay. 2006. Mapping protease substrates by using a biotinylated phage substrate library.. https://doi.org/10.1002/cbic.200500427

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