Elaine R. Mardis, Ph.D Read more about this drug ., Li Ding, Ph.D., David J. Dooling, Ph.D., David E. Larson, Ph.D., Michael D. McLellan, B.S., Ken Chen, Ph.D., Daniel C. Koboldt, M.S., Robert S. Fulton, M.S., Kim D. Delehaunty, B.A., Sean D. McGrath, M.S., Lucinda A. Fulton, M.S., Devin P. Locke, Ph.D., Vincent J. Magrini, Ph.D., Rachel M. Abbott, B.S., Tammi L. Vickery, B.S., Jerry S. Reed, M.S., Jody S. Robinson, M.S., Todd Wylie, B.S., Scott M. Smith, Lynn Carmichael, B.S., James M. Eldred, Christopher C. Harris, B.S., Jason Walker, B.A., B.S., Joshua B. Peck, M.B.A., Feiyu Du, M.S., Adam F. Dukes, B.A., Gabriel E. Sanderson, B.S., Anthony M. Brummett, Eric Clark, Joshua F. McMichael, B.S., Rick J. Meyer, M.S., Jonathan K. Schindler, B.S., B.A., Craig S. Pohl, M.S., John W.

Assay of HTRA1 Protease Activity To express HTRA1 in Escherichia coli as fusions with glutathione S-transferase, we subcloned wild-type or mutant HTRA1 complementary DNA , lacking codons 1 through 140, into the vector pGEX 6P-3 . The N-terminus of HTRA1 is definitely toxic to E. Coli. Amino acid substitution of the serine protease motif S328A, which abolishes the protease activity in HTRA1, was used as a negative control.14 Glutathione S-transferase fusion proteins had been overexpressed and purified. To get rid of the chance that a deletion of the N-terminus in HTRA1 impacts protease activity, we also performed exactly the same protease assay using the serum-free medium made up of cells stably expressing full-length wild-type or mutant HTRA1 tagged with a green fluorescent protein at the C-terminus.