Asmid was prepared from clone #8, and the inserted fragment (about 5 kbp

Asmid was prepared from clone #8, and the inserted fragment (about 5 kbp) was sequenced. As shown in Fig. 1B, 7 open reading frames (ORFs), among which 5 were full-length and 2 were truncated, were identified. Therefore, we cloned these 5 full-length ORFs individually into the pET21a expression vector, to determine which ORF is responsible for the degradation of ssDNA. As shown in Fig. 1C, the heat extract of the E. coli transformant containing PF2046 clearly expressed the nuclease activity.Figure 1. Screening and identification of the gene responsible for the DNA cleavage activity. (A) The E. coli cells, transformed with pUC118 containing each PstI-digested DNA fragment from the positive cosmid clone, were heated. The heat-stable cells extracts from each clone were assayed for DNase activity, using 32P-labeled d27 as the substrate. The reactions were analyzed by PAGE on a 12 gel containing 8 M urea followed by autoradiography. Lane M shows the GA ladder of d49R, generated by the Maxam-Gilbert reaction. The other lanes (1?2) are labeled d27 treated with heat-stable cell extracts from different clones. (B) The ORFs identified on the PstI-DNA fragment obtained from the positive clone #8. (C) The 5 full-length ORFs on the PstI fragment in the positive clone were individually cloned and expressed in E. coli, and the same DNase assay was performed using 32 P-labeled d49R as the substrate. PF2046 was identified as the gene responsible for the DNA cleavage activity. doi:10.1371/journal.pone.0058497.gmolecular mass of MedChemExpress PD-168393 PfuExo I is 25,596, as described above, the gel filtration results suggested that PfuExo I exists in a homotrimeric form in solution.Purification of the Recombinant Protein Encoded by PFThe recombinant protein encoded by PF2046 was successfully overproduced, by cultivating the E. coli cells bearing the expression plasmid, pPF2046, as described above, with IPTG induction. The protein was purified to near homogeneity (Fig. 2A) by the three sequential chromatography steps described in the Materials and Methods section. From a one liter culture (3.4 g cells), 1.5 mg of homogeneous protein was obtained. The relative molecular mass of the purified protein determined from its migration position in the SDS-PAGE gel corresponded to 25,596, calculated from the deduced amino acid sequence. We designated 10457188 the protein as PfuExo I.Detection of PfuExo I in the P. furiosus CellsUsing the highly purified PfuExo I protein, a polyclonal antibody was prepared. A western blotting analysis revealed that a protein band corresponding to the size of the recombinant PfuExo I (25,596), which specifically reacted with the antibody, was detected in the cell extract prepared from an exponential phase P. furiosus cell culture (Fig. 3A). This band was not detected in the cell extract prepared from the stationary phase culture of the cells, probably because of physiological proteolysis. The smaller bands, in addition to the main band, detected in the exponential phase may indicate that the function of PfuExo I is regulated with relatively short turnover in the P. furiosus cells. The buy Oltipraz appearance of an immunoreactive band at around 80,000 (Fig. 3A) is consistent with the results from the SDS-PAGE of the recombinant PfuExo I without heat treatment (Fig. 3B). The protein at 80,000 is also likely to be PfuExo I that was not completely denatured. The trimeric structure of this protein should be extremely stable, even in the presence of SDS.The Oligomeric State of PfuExo.Asmid was prepared from clone #8, and the inserted fragment (about 5 kbp) was sequenced. As shown in Fig. 1B, 7 open reading frames (ORFs), among which 5 were full-length and 2 were truncated, were identified. Therefore, we cloned these 5 full-length ORFs individually into the pET21a expression vector, to determine which ORF is responsible for the degradation of ssDNA. As shown in Fig. 1C, the heat extract of the E. coli transformant containing PF2046 clearly expressed the nuclease activity.Figure 1. Screening and identification of the gene responsible for the DNA cleavage activity. (A) The E. coli cells, transformed with pUC118 containing each PstI-digested DNA fragment from the positive cosmid clone, were heated. The heat-stable cells extracts from each clone were assayed for DNase activity, using 32P-labeled d27 as the substrate. The reactions were analyzed by PAGE on a 12 gel containing 8 M urea followed by autoradiography. Lane M shows the GA ladder of d49R, generated by the Maxam-Gilbert reaction. The other lanes (1?2) are labeled d27 treated with heat-stable cell extracts from different clones. (B) The ORFs identified on the PstI-DNA fragment obtained from the positive clone #8. (C) The 5 full-length ORFs on the PstI fragment in the positive clone were individually cloned and expressed in E. coli, and the same DNase assay was performed using 32 P-labeled d49R as the substrate. PF2046 was identified as the gene responsible for the DNA cleavage activity. doi:10.1371/journal.pone.0058497.gmolecular mass of PfuExo I is 25,596, as described above, the gel filtration results suggested that PfuExo I exists in a homotrimeric form in solution.Purification of the Recombinant Protein Encoded by PFThe recombinant protein encoded by PF2046 was successfully overproduced, by cultivating the E. coli cells bearing the expression plasmid, pPF2046, as described above, with IPTG induction. The protein was purified to near homogeneity (Fig. 2A) by the three sequential chromatography steps described in the Materials and Methods section. From a one liter culture (3.4 g cells), 1.5 mg of homogeneous protein was obtained. The relative molecular mass of the purified protein determined from its migration position in the SDS-PAGE gel corresponded to 25,596, calculated from the deduced amino acid sequence. We designated 10457188 the protein as PfuExo I.Detection of PfuExo I in the P. furiosus CellsUsing the highly purified PfuExo I protein, a polyclonal antibody was prepared. A western blotting analysis revealed that a protein band corresponding to the size of the recombinant PfuExo I (25,596), which specifically reacted with the antibody, was detected in the cell extract prepared from an exponential phase P. furiosus cell culture (Fig. 3A). This band was not detected in the cell extract prepared from the stationary phase culture of the cells, probably because of physiological proteolysis. The smaller bands, in addition to the main band, detected in the exponential phase may indicate that the function of PfuExo I is regulated with relatively short turnover in the P. furiosus cells. The appearance of an immunoreactive band at around 80,000 (Fig. 3A) is consistent with the results from the SDS-PAGE of the recombinant PfuExo I without heat treatment (Fig. 3B). The protein at 80,000 is also likely to be PfuExo I that was not completely denatured. The trimeric structure of this protein should be extremely stable, even in the presence of SDS.The Oligomeric State of PfuExo.

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