Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.
The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.
Cambon-Bonavita
M A
MA
Laboratoire de Caractérisation des Microorganismes Marins, DRV/VP, Ifremer centre de Brest, Plouzané, France.
Schmitt
P
P
Zieger
M
M
Flaman
J M
JM
Lesongeur
F
F
Raguénès
G
G
Bindel
D
D
Frisch
N
N
Lakkis
Z
Z
Dupret
D
D
Barbier
G
G
Quérellou
J
J
D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli; D005092:Exonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D019154:Protein Splicing; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D004256:DNA Polymerase I; D005092:Exonucleases; D008274:Magnesium