ISSN:
1433-4909
Keywords:
Key words Archaea
;
Thermococcus fumicolans
;
DNA polymerase
;
Exonuclease
;
Intein
;
Thermostability
;
Fidelity
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract 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°C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170 kDa 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/PL00010714
Permalink