ABSTRACT
A family B DNA polymerase gene from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I was highly expressed under the control of T7lac promoter of pET-28ARG in Escherichia coli BL21-CodonPlus(DE3)-RIL cells. The produced I. hospitalis (Iho) DNA polymerase was purified by heat treatment followed by HisTrap™ HP column and HiTrap™ SP column chromatographies. The molecular mass of the purified Iho DNA polymerase was 88 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH for Iho DNA polymerase activity was 7.0 and the optimal temperature was 70 °C. Iho DNA polymerase was strongly activated by the presence of magnesium ion at an optimum concentration of 3 mM. The optimal concentration of KCl for Iho DNA polymerase activity was 60 mM. The half-life of the enzyme at 94 °C was about 2 h. The optimal conditions for polymerase chain reaction (PCR) were determined. Iho DNA polymerase possesses 3'â5' exonuclease activity, and the fidelity of the Iho DNA polymerase was similar to that of Pfu and Vent DNA polymerases. However, Iho DNA polymerase provided more enhanced efficiency of PCR amplification than Pfu and Vent DNA polymerases. Iho DNA polymerase could successfully amplify a 2-kb λ DNA target with a 10-s extension time and could amplify a DNA fragment up to 8 kb λ DNA.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Desulfurococcaceae/enzymology , Polymerase Chain Reaction/methods , Amino Acid Sequence , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/isolation & purification , Exonucleases/metabolism , Hot Temperature , Sequence AlignmentABSTRACT
We previously reported that Tpa-S DNA polymerase (constructed via fusion of the Sso7d DNA binding protein to the C-terminus of Thermococcus pacificus (Tpa) DNA polymerase) is more efficient in long and rapid PCR than wild-type Tpa, Taq, or Pfu DNA polymerases. However, Tpa-S DNA polymerase had a low yield of PCR products compared with commercialized Taq or Pfu DNA polymerases. To improve the yield of PCR products, mutant Tpa-S DNA polymerases were created via site-directed mutagenesis. In this study, we have targeted the N213 residue in the Exo II motif and the K501 residue in the Pol III motif. The mutant Tpa-S DNA polymerases showed enhanced PCR yields compared to that of the Tpa-S DNA polymerase. Specifically, the double mutant Tpa-S N213D/K501R DNA polymerase had an approximately three-fold increase in the yield of 8-10kb PCR products over that of the Tpa-S DNA polymerase, and catalyzed amplification of a 12kb PCR product using a lambda template with an extension time of 30s. Even though the mutation is in the Exo II motif, the error rate of the double mutant Tpa-S N213D/K501R (2.79×10(-5)) was nearly the same as that seen in the Pfu DNA polymerase (2.70×10(-5)).
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Polymerase Chain Reaction/methods , Thermococcus/enzymology , Thermococcus/genetics , Cloning, Molecular , DNA/chemistry , DNA/metabolism , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Escherichia coli/genetics , Hydrogen-Ion Concentration , Kinetics , Magnesium Chloride/chemistry , Mutation , Plasmids/chemistry , Plasmids/metabolism , Potassium Chloride/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
The aims of this study were to investigate the prevalence of atopy, asthma, and obesity in university freshmen and to determine whether leptin is associated with bronchodilator reversibility in obesity. A total of 537 university freshmen completed International Study of Asthma and Allergies in Children (ISAAC) questionnaire and underwent skin prick testing and bronchodilator reversibility test. The prevalences of asthma, wheeze, and atopy were 10 (1.9%), 84 (15.6%), and 198 (36.9%), respectively. The mean (+/- SD) bronchodilator response (5.1 +/- 4.4%) was higher in the overweight/obese men than in the normal male subjects (3.7 +/- 3.2%, p < 0.05). The mean leptin level in the overweight/obese men was 5.55 +/- 3.48 ng/mL, which was significantly higher than that (2.78 +/- 1.65 ng/mL) of the normal male subjects. The prevalence of asthma among university freshmen is seriously under-diagnosed and leptin may play a role in bronchodilator reversibility in overweight/obese young men.
