Mapping protease substrates by using a biotinylated phage substrate library.
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.
Scholle
Michael D
MD
Combinatorial Biology Unit, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Kriplani
Ushma
U
Pabon
Amanda
A
Sishtla
Kamakshi
K
Glucksman
Marc J
MJ
Kay
Brian K
BK
D017104:Bacteriophage M13 / Q000737:chemistry; D019921:Biotinylation; D015951:Factor Xa / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008666:Metalloendopeptidases / Q000737:chemistry; D010447:Peptide Hydrolases / Q000737:chemistry*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D012680:Sensitivity and Specificity
D019151:Peptide Library; D010455:Peptides; D010447:Peptide Hydrolases; D015951:Factor Xa; D008666:Metalloendopeptidases; C062316:neurolysin