Characterization of a randomized FRET library for protease specificity determination

Abstract

Protease specificity determination is an important first step when characterizing novel proteases. Given the large number of proteases that are known to exist from genomic sequencing efforts, we reason 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 using the fluorescent proteins GFP and DsRed for such a purpose. Using a DNA “cassette” that allowed for directional insertion of annealed oligonucleotides between the genes encoding the GFP and DsRed proteins, we constructed a library using a mixture of standard nucleotide bases at 27 positions in the center of the oligonucleotide cassette. This resulted in a randomized linker region between these fluorescent donor–acceptor pairs to produce substrates with varied amino acids located between the proteins. Kinetic assays were then performed and monitored using the increase in GFP fluorescence to arrive at relative reaction velocities for a set of enzymes. These results demonstrated the ability of the enzymes tested to discriminate between different substrates and the resistance of GFP and DsRed to proteolysis. Colony screening, using color development and restriction enzyme digests, were shown to help eliminate DNA samples in the library that contained stop codons and/or deletions and a flow plan for the efficient use of the library is presented.