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Development of a high throughput and generally applicable screening method for investigating protease/substrate interaction
Author(s) -
Bae Weon,
Zhou Jian Hong,
Graves Steven W
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a50-d
Subject(s) - substrate (aquarium) , high throughput screening , throughput , protease , computational biology , chemistry , computer science , nanotechnology , biology , materials science , biochemistry , enzyme , telecommunications , ecology , wireless
Proteases regulate many diverse biological pathways and are also common components of many bacterial toxins targeting cell signaling pathways. Consequently they become attractive targets for pharmaceuticals and detection assays. However, the study of proteases and development of inhibitors for toxin proteases does not follow a single established methodology. In order to develop a high‐throughput and generally applicable method for investigating protease/substrate interaction, we developed a flow cytometry‐based assay system combined with cell surface display. We constructed both a bacterial and a yeast surface display systems, which express a fluorescing reporter protein fused to either a short cleavage site or a full length substrate of the Lethal Factor metalloprotease (LF) of Bacillus anthracis. First, we constructed a yeast surface display system expressing a fusion protein containing a LF cleavage site, LF15 or CS19, which were known as consensus sequences for a LF cleavage. LF15 was superior to CS19. Tandem repeat of LF15 and CS19 did not significantly improve the cleavage. Second, we developed an E.coli surface display system, which express either a full length of mitogen activated protein kinase kinase 2 (MAPKK2), which is one of natural substrates for LF, or only LF15. Interestingly, a full length of substrate was shown to be superior to a short cleavage sequence only. Because most of the screening methods use a library of small peptides, our results showed the significance of studying the intact substrate protein. We are currently constructing a mutation library both of the small conserved residues and of the full length to screen them to identify better substrate for the LF