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Zappa, Sebastien; Rolland, Jean-luc; Flament, Didier; Gueguen, Yannick; Boudrant, Joseph; Dietrich, Jacques. |
This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70 degreesC, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105 degreesC of 18 and 5 h, respectively.... |
Tipo: Text |
Palavras-chave: Escherichia coli; Characterization; Isolation; Gene; Alkaline phosphatase; Pyrococcus abyssi; Archaeon. |
Ano: 2001 |
URL: http://archimer.ifremer.fr/doc/2001/publication-1268.pdf |
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Henneke, Ghislaine; Gueguen, Yannick; Flament, Didier; Azam, Philippe; Querellou, Joel; Dietrich, Jacques; Hubscher, Ulrich; Raffin, Jean-paul. |
The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (Pab RFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the Pab RFC-small subunit could be purified, while the large subunit (Pab RFC-large) alone was completely insoluble. The highly purified Pab RFC complex possessed an... |
Tipo: Text |
Palavras-chave: PCNA binding domain; Pyrococcus abyssi; Hyperthermophile; Archaea; Replication factor C. |
Ano: 2002 |
URL: https://archimer.ifremer.fr/doc/2002/publication-675.pdf |
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Henneke, Ghislaine; Raffin, Jean-paul; Ferrari, Elena; Jonsson, Zophonías; Dietrich, Jacques; Hubscher, Ulrich. |
We have cloned the gene encoding proliferating cell nuclear antigen (PCNA) from the hyperthermophilic euryarchaeote Thermococcus fumicolans (Tfu). Tfu PCNA contains 250 amino acids with a calculated M-r of 28,000 and is 26% identical to human PCNA. Next, Tfu PCNA was overexpressed in Escherichia coli and it showed an apparent molecular mass of 33.5 kDa. The purified Tfu PCNA was tested first with recombinant Tfu DNA polymerase I (Tfu pol) and second with calf thymus DNA polymerase delta (pol delta). When tested with the homologous Tfu pol on bacteriophage lambda DNA, large amounts of Tfu PCNA were required to obtain two- to threefold stimulation. Surprisingly, however, Tfu PCNA was much more efficient than human PCNA in stimulating calf thymus pol delta.... |
Tipo: Text |
Palavras-chave: DNA polymerase delta; Conservation; Thermococcus fumicolans; Archaea; Hyperthermophile; PCNA. |
Ano: 2000 |
URL: http://archimer.ifremer.fr/doc/2000/publication-659.pdf |
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