, 50°C for 45 sec, and 72°C for 45 sec. Amplified fragments were cloned into pET101/D-TOPO vector and sequenced to determine if the glutamic acid (E) at position 49 was replaced by alanine (A). The resulting recombinant plasmid was designed as pSTM0551E49A-His. Further protein induction and purification were performed using the same procedure as for STM0551-His fusion protein. Similarly FimY-His fusion protein was Idasanutlin constructed using fimY-TOPO-F and fimY-TOPO-R primers. PDE activity assay In vitro PDE activity
assays were performed using purified STM0551-His, STM0551E49A-His and FimY-His proteins. Test protein was suspended in the assay buffer (50 mM Tris–HCl and 1 mM MnCl2, pH 8.5) supplemented with 5 mM bis (p-nitrophenol) phosphate (bis-pNPP) as previously described
[40, 41]. Reactions were incubated at 37°C overnight. The release of p-nitrophenol was quantified at OD410 in a spectrophotometer (WPA Biowave II, Cambridge, UK). Statistical analysis All statistical data were analyzed using Student’s t-test. Differences in measurements with a p value of < 0.05 were considered to be significant. Acknowledgements This study was supported by the National Science Council, Taiwan under contract no. NSC98-2313-B-038-001-MY3. We would like to thank Dr. Ching-Hao Teng from National Cheng-Kung University, Taiwan for providing selleck kinase inhibitor pKD46 and pKD13 plasmids. We would also like to thank Ms. S.-T. Kuo from the Animal Health Research Institute, Council of Agriculture, Dichloromethane dehalogenase Taiwan for assistance
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