Pyrophosphatase ppiase

SAM, SAH, 5′-GMP, Tris-base, β-mercaptoethanol (β-Me), KCl and the reagents used to optimize crystallization conditions were purchased from Sigma–Aldrich (St Louis, MO, USA). MgCl₂, NaCl, adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate were from Sangon Biotech (Shanghai, China). [Methyl-³H] SAM was obtained from PerkinElmer (Waltham, MA, USA); crystallization kits were from Hampton research (Aliso Viejo, CA, USA). Primers for polymerase chain reaction were synthesized by Invitrogen (Shanghai, China) and Biosune (Shanghai, China); the pET22b vector was from Merck–Millipore (Darmstadt, Germany). The KOD-plus mutagenesis kit, Pyrobest DNA polymerase and dNTP mixture were obtained from Takara (Shiga, Japan); T4 ligase and other restriction endonucleases were obtained from MBI Fermentas (Pittsburgh, PA, USA). Pyrophosphatase (PP_iase) was purchased from Roche Applied Science (Basel, Switzerland). Ni²⁺-NTA Superflow was obtained from Qiagen (Dusseldorf, Germany). The Superdex 200 column and 3 mm filter papers were from GE Healthcare (Fairfield, CT, USA). The analog of SAM, sinefungin, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

l-Thr, l-Ser, dithiothreitol, nucleoside triphosphates (NTPs), guanosine monophosphate (GMP), tetrasodium pyrophosphate, Tris-base, MgCl₂, NaCl and inorganic pyrophosphate were purchased from Sigma (St Louis, MO, USA). [¹⁴C]Thr was obtained from Biotrend Chemicals (Destin, FL, USA). [α-³²P]ATP and [γ-³²P]ATP were obtained from Perkin Elmer Inc. (Waltham, MA, USA). T4 DNA ligase, T4 PNK (polynucleotide kinase), RNase T1, RNase S1, and restriction endonucleases were obtained from Thermo Scientific (Pittsburgh, PA, USA). Phusion high-fidelity DNA polymerase was purchased from New England Biolabs (Ipswich, MA, USA). Ni²⁺-NTA (nitrilotriacetic acid) Superflow was purchased from Qiagen Inc. (Germany). Pyrophosphatase (PPiase) was obtained from Roche Applied Science (China). The dNTP mixture was purchased from Takara (Japan). Oligonucleotide primers were synthesized by Biosune (China). Escherichia coli Rosetta (DE3) cells were purchased from Stratagene (Santa Clara, CA, USA).