Ubiquitination Project in active screening mode at Meso Scale Discovery (Gaithersburg). Approximately 90,000 (out of ~120,0000) extracts so far screened. Hit rates for primary assay 1.5-1.8%, secondary multiplexed screen (MDM2, XIAP, RNF28, Nedd4) for selectivity reduces hit rate ~50%. All primary and secondary screening should be complete by the end of October. Biotinylated VCP and its truncated mutants were provided from Li lab. Using these materials, we are currently screening a peptide library. IGF1R Large-scale phosphorylation of IGF1R was successful. Large-scale phosphorylation of IR resulted in the cytosolic construct crashing out of solution as a precipitate. Additional 38 mg of IR has been produced and purified. Nissley lab will work (on small scale) to optimize phosphorylation of IR to prevent precipitation. We have received receptor binding proteins 14-3-3 sigma, 14-3-3 beta, GIPC, and LARG. The other binding proteins (Shc, IRS-1, truncated IRS-1, RACK) had varying degrees of problems with expression/purification. Full report from the PEL is expected by the end of the week. Meeting with PEL, Nissley lab and MTDP to be scheduled the following week. Smad3/4 Meeting scheduled for October 7th with Anita Roberts and MTDP project team to discuss latest developments. CyclinA/CDK2 Still waiting on the 800-member spiroketal library from Porco lab to begin small-scale screen for inhibition of protein-protein interactions. Phage-displayed peptide library Although we have successfully made one peptide library this spring, we found the original procedure to construct the first library did not work for other library construction. Therefore, during this summer we spent our time to develop a method to simplify the construction process and improve the diversity of peptide libraries. As the results, we have now reproducible and simple method to construct a series of phage-displayed combinatorial peptide libraries that possess diverse complexities and unique structures. We were also able to increase the ligation efficiency 100-1000 times. We expect to construct 5-10 libraries by the end of this year. Kai1 We finally able to make a paramagnetic proteoliposomes in which the intracellular side of Kai1 is located outside of artificial lipid membrane and the extracellular side of it is located inside of the lipid bilayer. Dr. Tarasova synthesized two peptide fragments of Kai1; one represent a part of 2nd extracellular domain of Kai1 and the other represent the C-terminal intracellular tail of Kai1. She subsequently attached the peptides on magnetic beads. Using these materials, we are currently screening a peptide library. STEAP Tarasova lab provided us the synthesized peptide that represents the extracellular domain of STEAP on magnetic beads. Using this material, we are currently screening a peptide library. Death receptor, EMAP Based on our suggestion, Lipkowitz and Libutti labs have been working on construction/expression/purification of DR4/5 intracellular domain protein and the biotinylated EMAP protein, respectively. Still waiting to obtain the highly pure target molecule. Human scFv Library Human scFv library was acquired from MRC to isolate scFv against target of interest in CCR investigators. Nuclear Receptors Preliminary experiments on cells expressing GFP-ER conjugates have clarified issues/requirements for nuclear translocation imaging assays. The cell lines tested to date have not had sufficiently bright fluorescence to allow for establishment of a reliable, automated, image-based translocation assay based on standard screening assay criteria (e.g. Z-factors, signal/background ratio, CVs, etc.). Alternative, apparently brighter, cell lines are being expanded in Gordon Hager's lab. These are expected to be available for testing in October. If acceptable, methods development will follow. RNaseH Primary screening and confirmation of all pure compound libraries and extracts (>200,000 samples) has been completed. Secondary evaluation of pure compounds is complete, and extract prioritization is well underway. A meeting has been scheduled (Oct.10) to discuss medicinal chemistry of hits with the University of Pittsburgh group. Extracts will be chosen for isolation projects once prioritization has been completed. DNA Methyl Transferase We have attempted to use the ability of a protein, MeCP2, which binds to methylated DNA, to capture the product of DNMT1 enzymatic action on a fluorescently labeled DNA substrate. While the MeCP2-GST fusion protein bound a methylated fluorescently labeled oligo, as measured by a gel shift assay and a fluorescence polarization assay, we have been unable to capture the oligo on the surface of either high protein binding or glutathione plates. Other alternative formats such as a biotin capture or capillary electrophoresis are under consideration. GPCR Awaiting resolution of deliberations on acquisition of Norak technology; application of cell standardization studies to this project. Suresh Arya/Geetanjali Sachdeva Evaluation of expression of GFP in HeLa cells completed. Assessment by fluorescent microscopy and Discovery 1 imaging with standardization of readout by incorporation of multiplexed stain to assess cell number. Studies have resulted in acquisition of cells with bright GFP expression as a positive control for other imaging studies.
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