Was added to 0.15 (w/v). The solution was stirred on ice

Was added to 0.15 (w/v). The solution was eFT508 web stirred on ice for 30 min, and centrifuged for 10 min at 23,7006g. Ammonium sulfate was added to the supernatant to 80 saturation, and the solution was stirred on ice for 1 hour. The precipitate from the ammonium sulfate treatment was suspended in 8 ml of buffer A containing 1 M ammonium sulfate, and applied to a hydrophobic column (HiTrap phenyl, GE Healthcare UK Ltd), which was developed with a linear gradient of 1.0? M ammonium sulfate. The PfuExo I-containing fractions were applied to an anion-exchange column (HiTrapQ, GE Healthcare UK Ltd), after dialysis against buffer A containing 0.1 M NaCl. The column was developed with a 0.1?.0 M NaCl gradient. The active fraction eluted at 0.15 M NaCl was diluted to 0.015 M NaCl, and applied to an affinity column (HiTrap heparin, GE Healthcare UK Ltd), which was developed with a linear gradient of 0.1?.8 M NaCl. The eluted fractions were concentrated and stored at 4uC. The protein concentrations were determined by measuring the absorbance at 280 nm, with an extinction coefficient of 35,410 M21 cm21, which was obtained by the method described earlier [44].Exonuclease AssayThe heat-stable protein extracts or purified PfuExo I was incubated with 5 nM of various 32P-labeled substrate DNAs in a standard reaction mixture, containing 20 mM Tris-HCl (pH 8.8), 5 mM MgCl2, 50 mM NaCl, and 0.1 mg/ml BSA. Reaction time and temperature are described in each figure legend. The reactions were stopped with an equal volume of deionized formamide. These samples were subjected to polyacrylamide8 M urea gel electrophoresis, and the degradation products were visualized by autoradiography.Cloning and Identification of the PfuExo I GeneThe cosmid-based genomic library, in which each of clone contains the P. furiosus DNA fragment with 35?0 kbp, was prepared as described previously [24]. Then, heat-stable protein extracts were obtained from 500 independent clones by the heat treatment at 80uC for 10 min. The heat-stable extracts were used for the screening of DNase activity. Cosmid DNA was prepared from the clone exhibiting the heat-stable DNase activity, and was partially digested by PstI. The DNA fragments were inserted into pUC118, and the resultant plasmids were introduced into E. coli JM109. The heat-stable extracts were prepared from each clone, and were used for the 25331948 DNase assay. The DNA fragment from the positive clone was EAI045 site analyzed by DNA sequencing, which revealed the presence of 5 full-length ORFs in the 5-kbp fragment. These 5 ORFs were amplified by PCR directly from the genomic DNA of P. furiosus. Each amplified gene was cloned into the pGEM-T easy vector (Promega). The cloned genes were digested by either NdeI-BamHI or NcoI-BamHI, and were inserted into the corresponding sites of pET21a or pET21d. The constructed plasmids were designated as pPF2045, pPF2046, pPF2047, pPF2048, and pPF2049, respectively. E. coli cells were transformed with the plasmids, and heat-stable extracts were prepared from each clone and used for the DNase assay.Endonuclease ActivityPurified PfuExo I (100 nM) was added to the reaction mixture containing 5 ng/ml of pBR322 or M13 mp18 ssDNA (Takarabio), in the standard reaction condition for the exonuclease assay for 30 min at 65uC. The reactions were terminated by the addition of an equal volume of phenol. The reaction products were fractionated by electrophoresis on a 1 agarose gel, and the DNA was visualized by ethidium bromide staining.W.Was added to 0.15 (w/v). The solution was stirred on ice for 30 min, and centrifuged for 10 min at 23,7006g. Ammonium sulfate was added to the supernatant to 80 saturation, and the solution was stirred on ice for 1 hour. The precipitate from the ammonium sulfate treatment was suspended in 8 ml of buffer A containing 1 M ammonium sulfate, and applied to a hydrophobic column (HiTrap phenyl, GE Healthcare UK Ltd), which was developed with a linear gradient of 1.0? M ammonium sulfate. The PfuExo I-containing fractions were applied to an anion-exchange column (HiTrapQ, GE Healthcare UK Ltd), after dialysis against buffer A containing 0.1 M NaCl. The column was developed with a 0.1?.0 M NaCl gradient. The active fraction eluted at 0.15 M NaCl was diluted to 0.015 M NaCl, and applied to an affinity column (HiTrap heparin, GE Healthcare UK Ltd), which was developed with a linear gradient of 0.1?.8 M NaCl. The eluted fractions were concentrated and stored at 4uC. The protein concentrations were determined by measuring the absorbance at 280 nm, with an extinction coefficient of 35,410 M21 cm21, which was obtained by the method described earlier [44].Exonuclease AssayThe heat-stable protein extracts or purified PfuExo I was incubated with 5 nM of various 32P-labeled substrate DNAs in a standard reaction mixture, containing 20 mM Tris-HCl (pH 8.8), 5 mM MgCl2, 50 mM NaCl, and 0.1 mg/ml BSA. Reaction time and temperature are described in each figure legend. The reactions were stopped with an equal volume of deionized formamide. These samples were subjected to polyacrylamide8 M urea gel electrophoresis, and the degradation products were visualized by autoradiography.Cloning and Identification of the PfuExo I GeneThe cosmid-based genomic library, in which each of clone contains the P. furiosus DNA fragment with 35?0 kbp, was prepared as described previously [24]. Then, heat-stable protein extracts were obtained from 500 independent clones by the heat treatment at 80uC for 10 min. The heat-stable extracts were used for the screening of DNase activity. Cosmid DNA was prepared from the clone exhibiting the heat-stable DNase activity, and was partially digested by PstI. The DNA fragments were inserted into pUC118, and the resultant plasmids were introduced into E. coli JM109. The heat-stable extracts were prepared from each clone, and were used for the 25331948 DNase assay. The DNA fragment from the positive clone was analyzed by DNA sequencing, which revealed the presence of 5 full-length ORFs in the 5-kbp fragment. These 5 ORFs were amplified by PCR directly from the genomic DNA of P. furiosus. Each amplified gene was cloned into the pGEM-T easy vector (Promega). The cloned genes were digested by either NdeI-BamHI or NcoI-BamHI, and were inserted into the corresponding sites of pET21a or pET21d. The constructed plasmids were designated as pPF2045, pPF2046, pPF2047, pPF2048, and pPF2049, respectively. E. coli cells were transformed with the plasmids, and heat-stable extracts were prepared from each clone and used for the DNase assay.Endonuclease ActivityPurified PfuExo I (100 nM) was added to the reaction mixture containing 5 ng/ml of pBR322 or M13 mp18 ssDNA (Takarabio), in the standard reaction condition for the exonuclease assay for 30 min at 65uC. The reactions were terminated by the addition of an equal volume of phenol. The reaction products were fractionated by electrophoresis on a 1 agarose gel, and the DNA was visualized by ethidium bromide staining.W.