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Mapping Protease Substrates by Using a Biotinylated Phage Substrate Library
Author(s) -
Scholle Michael D.,
Kriplani Ushma,
Pabon Amanda,
Sishtla Kamakshi,
Glucksman Marc J.,
Kay Brian K.
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500427
Subject(s) - biotinylation , streptavidin , protease , peptide , capsid , chemistry , cleavage (geology) , peptide library , substrate (aquarium) , scissile bond , biotin , bacteriophage , biochemistry , combinatorial chemistry , escherichia coli , biology , peptide sequence , enzyme , gene , paleontology , ecology , fracture (geology)
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 (≥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.