(623w) Protein Engineering for High-Throughput Screening and Protease Dynamic Analysis

Designing simple and sensitive enzymatic analysis methods has been of great interest for enzymatic kinetic assay and high-throughput screening (HTS).  The need to measure multiple protease activities simultaneously has resulted in the invention of multiplex microsphere based protease assays. Previously, we have described the development of a microsphere based assay for the proteases Factor Xa, Bacillus anthracis Lethal Factor (LF), and Clostridium botulinum type A light chain (BoNT/A LC) via high throughput flow cytometry. The selection of potential inhibitors to these proteases is of great importance due to their key roles in the lethality of anthrax and the toxicity of BoNT. This screening platform uses suspension microsphere arrays coated with streptavidin and multiplexed via different levels of intrinsic microsphere fluorescence. The fluorescent fusion protein, which contains a biotinylation tag and a GFP domain separated by the full-length protein substrate, is bound to the microsphere and protease activity is detected via loss of fluorescence from the microsphere surface by flow cytometry. Here we present the development of a multiplex substrate set that has been engineered for use with three proteases (BoNT/A, F LCs and LF) simultaneously. These substrates have been attached to a four-plex microsphere set and mixed with the proteases that were arrayed in 1536-well plates containing the >1100 compounds of the Prestwick library. We have demonstrated multiplex microsphere based protease assays analyzed by flow cytometry and high throughput flow cytometry based screening that discovered several interesting inhibitors to the proteases. We will also present confirmatory assays and dose response curves for the identified compounds. The microsphere based flow cytometry assay is a promising approach with several advantages including: low substrate costs, small reaction volumes, homogenous assay format, and use of full length substrates to detect all pertinent interactions; however, it has limitations in collection of traditional kinetic data. Therefore, we have developed several novel methods to study the protease/substrate kinetics that enable determination of key kinetic constants of protease activities. One such method uses fluorescence resonance energy transfer (FRET) between a full length protease substrate and a fluorescently labeled streptavidin protein to determine the solution based enzymatic activities. In this case, we used biotinylation tag and GFP labeled protein substrates connected to streptavidin-Cy3 conjugate as the cleavage targets for proteases in solution. Based on the FRET from GFP to Cy3, the fluorescence intensity change was recorded on a fluorometer or a plate reader. Additionally, the development of another microsphere assay based on fusion protein substrates and engineered biomimetic lipid bilayer microsphere surfaces will be presented. It is capable of screening several different matrix metalloproteases (MMPs) activities which are suggested to require substrates in lipid bilayers. In this study, a MMP substrate was designed with a GFP domain and a membrane insertion active tag. In summary, we present several new protease assays that utilize custom engineered fusion proteins that enable measurement of both surface based and full-length solution based protease assays. As such, these fusion proteins enable high-throughput inhibitor screening and detailed kinetic studies.