Cloning, expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR.
The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E. coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.
Dabrowski
Sławomir
S
The Environmental Microbiology and Biotechnology Research Group, BioCentrum, Søltofts Plads, Building 227, The Technical University of Denmark, DK-2800 Kgs., Lyngby, Denmark.
Kiaer Ahring
Birgitte
B
D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics / Q000378:metabolism; D019641:DNA, Archaeal / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D030561:Databases, Nucleic Acid; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine; D006358:Hot Temperature; D009842:Oligopeptides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011489:Protein Denaturation / Q000187:drug effects; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011755:Pyrophosphatases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry / Q000494:pharmacology
D019843:Archaeal Proteins; D003062:Codon; D019641:DNA, Archaeal; D003845:Deoxycytosine Nucleotides; D003856:Deoxyuracil Nucleotides; D011756:Diphosphates; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; C027078:deoxyuridine triphosphate; C024107:2'-deoxycytidine 5'-triphosphate; D012965:Sodium Chloride; D006639:Histidine; D004259:DNA-Directed DNA Polymerase; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase