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FRET Libraries for Protease Specificity Determination
Author(s) -
Selby Thomas Lee
Publication year - 2008
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.22.1_supplement.793.6
Subject(s) - proteases , protease , förster resonance energy transfer , proteolysis , peptide library , linker , serine protease , green fluorescent protein , computational biology , chemistry , genomic library , dna , biology , biochemistry , enzyme , peptide sequence , fluorescence , gene , base sequence , computer science , physics , quantum mechanics , operating system
Specificity determination is an important step in characterizing novel proteases. Given the large number of proteases that are known to exist from genomic sequencing efforts, reasons that sensitive, reliable, and high‐throughput methods to determine protease specificity must be developed. This study describes the construction and initial characterization of a protein based FRET library to rapidly determine protease specificity using GFP and DsRed. A randomized DNA linker region was inserted between these donor‐acceptor FRET proteins to produce a library of protease substrates with varied amino acid sequences. Kinetic assays were then performed by monitoring the increase in GFP fluorescence as a function of time and substrate concentration to determine reaction velocities and specificity for a set of proteases. Our results demonstrate the ability of the proteases to discriminate between different linker regions as well as the resistance of GFP and DsRed to proteolysis. Screening methods utilizing color development of individual E. coli colonies and restriction enzyme digests, were used to eliminate those DNA samples in the library that contained stop (or rare) codons and/or deletions and a flow plan for efficient use of the library in high throughput and single molecule screening is also presented.