Purification And Characterization Of Catalytic Domain Of PTP-LAR With Or Without D2 Domain For High-throughput Inhibitor Screening

Human leukocyte common antigen-related molecule (LAR), a transmembrane receptor-type protein tyrosine phosphatase containing two conserved phosphatase domains (D1 and D2), has been implicated as a physiological negative regulator of the insulin receptor and a potential drug; target for the treatment of type 2 diabetes and other associated metabolic syndromes. To establish a microplate based assay for high-throughput inhibitor screening, the high yielded active Dl and D1D2 were expressed in Escherichia coli as GST-fusion proteins and purified to homogeneity by affinity chromatography. Dl or D1D2 obtained from cleavage by thrombin is indistinguishable with GST-fusion Dl or D1D2 respectively in nearly all aspects, including pH optima, kinetic parameters on activity and vanadate inhibition. Dl is similar with to D1D2, but more active. A colormetric assay based on the cleavage of pNPP substrates by Dl was set up and a set of 33,600 compounds chemicals and extracts from Chinese National Compound Library were screened. Further analysis by using the hits from screening indicated there is a correlation between IC50 values on Dl and GST-D1 or D1D2. A novel competitive LMW PTP-LAR inhibitor was identified and showed distinct specificity compared with several other proteintyrosine phosphatases, FTP IB and CDC25A, B.