Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR.

The family B DNA polymerase gene from the euryarchaeon Thermococcus barophilus Ch5 (Tba5) contains an open reading frame of 6198 base pairs that encodes 2065 amino acid residues. The gene is split by three inteins that must be spliced out to form the mature DNA polymerase. A Tba5 DNA polymerase gene without inteins (genetically intein-spliced) was expressed under the control of the pET-28b(+)T7lac promoter in E. coli Rosetta 2(DE3)pLysS cells. The molecular mass of the purified Tba5 DNA polymerase was about 90kDa consistent with the 90,470Da molecular mass calculated based on the 776 amino acid sequence. The optimal pH for Tba5 DNA polymerase activity was 7.5 and the optimal temperature was 70-75°C. The enzyme possessed 3'→5' exonuclease activity and was activated by magnesium ions. PCR amplification using Tba5 DNA polymerase enables high-yield for 1- to 6-kb target DNA products, while 8- to 10-kb target DNA products were amplified at low or inefficient levels. To simultaneously improve product yield and amplification fidelity, Tba5 plus DNA polymerase mixtures were constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase. The Tba5 plus DNA polymerase mixtures robustly amplified up to 25-kb λ DNA fragments. In addition, the PCR error rate of Tba5 plus3 and Tba5 plus4 mixtures were much lower than those of wild-type Tba5 DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and Pfu plus DNA polymerase.

Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase.

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3×10(-5)) was roughly similar to that of Pfu DNA polymerase (4.8×10(-5)), but much lower than those of wild-type Neq DNA polymerase (57.2×10(-5)), Neq A523R DNA polymerase (13.1×10(-5)), and Neq N540R DNA polymerase (37.7×10(-5)). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.

Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination.

In 2013, Tomato chlorosis virus (ToCV) was identified in symptomatic tomato plants in Korea. In the present study, a loop-mediated isothermal amplification (LAMP) method was developed using four specific primers designed against ORF6 in ToCV RNA2 to detect ToCV rapidly and with high sensitivity. The optimized reaction involved incubation of a reaction mixture containing 2U Bst DNA polymerase and 4mM MgSO4 for 1h at 60-62 °C. Although specific and rapid detection of ToCV by LAMP was confirmed, false-positive reactions caused by carry-over contamination sometimes occurred because of the high sensitivity of LAMP compared with other detection methods. To prevent false-positive reactions, dUTP was substituted for dTTP and uracil-DNA glycosylase (UDG) was added to the LAMP reaction. First, the LAMP reaction was conducted successfully with substitution of dUTP for dTTP. Before the next reaction, LAMP products with incorporated dUTP were cleaved selectively by UDG without any effect on thymine-containing DNA (template DNA). This modified LAMP method complemented with UDG treatment to prevent carry-over contamination offers a potentially powerful method for detecting plant viruses.

Enhanced PCR efficiency of high-fidelity DNA polymerase from Thermococcus waiotapuensis.

Twa DNA polymerase from hyperthermophilic archaeon Thermococcus waiotapuensis has exceedingly high fidelity among family B DNA polymerases. However, Twa DNA polymerase has significant shortcomings in terms of a low extension rate and poor processivity. To resolve these weaknesses, we focused on two amino acid residues (N565 and H633) in the palm and thumb subdomains of the Twa DNA polymerase. These two residues were replaced by site-directed mutagenesis and the enzymatic properties of the mutants were analyzed. Here, Twa H633R DNA polymerase showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of processivity (2-fold), extension rate (1.5-fold) and PCR efficiency. Kinetic analysis using DNA as a template revealed that the kcat value of the Twa H633R mutant was similar to that of wild-type, but the Km of the Twa H633R mutant was about 1.6-fold lower than that of the wild-type. These results showed that the Arg residue substitution at H633 located in the thumb subdomain has a positive effect on processivity, extension rate and PCR efficiency, suggesting that the Twa H633R mutant allows a conformational change for easy access of the primer-template to the binding site of the polymerase domain.

Mutations in the palm subdomain of Twa DNA polymerase to enhance PCR efficiency and its function analysis.

Among the family B DNA polymerases, the Twa DNA polymerase from T. wiotapuensis, a hyperthermophilic archaeon, has exceedingly high fidelity. For applications in PCR, however, the enzyme is limited by its low extension rate and processivity. To resolve these weaknesses, we focused on two amino acid residues (A381 and N501) located at the palm subdomain of Twa DNA polymerase. Following replacement of these residues by site-directed mutagenesis, Twa N501R DNA polymerase showed significantly improved polymerase function compared to the wild-type enzyme in terms of processivity (3-fold), extension rate (2-fold) and PCR efficiency. Kinetic analysis using DNA as template revealed that the kcat value of the Twa N501R mutant was similar to that of wild-type, but the Km of the Twa N501R mutant was about 1.5-fold lower than that of the wild-type. These results suggest that a positive charge at residue 501 located in the forked-point does not impede catalytic activity of the polymerase domain but stabilizes interactions between the polymerase domain and the DNA template.

Characterization of a family B DNA polymerase from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I and its application to PCR.

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'→5' 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.

Enhancing the processivity of a family B-type DNA polymerase of Thermococcus onnurineus and application to long PCR.

Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalised to others. Amplification efficiency is lower in family B-type DNA polymerases than in family A-type (Taq) polymerases because of their strong 3'-5' exonuclease-activity. Here, we have red the exonuclease domain of the Thermococcus onnurineus NA1 (TNA1) DNA polymerase, especially Asn210 to Asp215 residues in Exo II motif (NXXXFD), to improve the processivity. N213D mutant protein had higher processivity and extension rate than the wild-type TNA1 DNA polymerase, retaining a lower mutation frequency than recombinant Taq DNA polymerase. Consequently, the N213D mutant could amplify target DNA up to 13.5 kb in length from human genomic DNA and 16.2 kb in length from human mitochondrial DNA while wild-type TNA1 amplified target DNA of 2.7 kb in length from human genomic DNA.

Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance.

The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.

Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis.

The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90 kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75 °C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99 °C was about 4 h. The optimal buffer for PCR with Twa DNA polymerase was 50 mM Tris-HCl (pH 8.2), 2.0 mM MgCl2, 30 mM KCl, 2.0 mM (NH4)2SO4, 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.

Characterization and PCR applications of dUTPase from the hyperthermophilic euryarchaeon Thermococcus pacificus.

We cloned and sequenced the gene encoding Thermococcus pacificus dUTPase (Tpa dUTPase). The Tpa dUTPase gene consists of 471 bp and encodes a 156-amino acid protein. The deduced amino acid sequence of Tpa dUTPase has high sequence similarity with other archaeal dUTPases. The Tpa dUTPase had an 18-kDa major protein band consistent with the 17,801 Da molecular mass calculated based on the amino acid sequence. The specific activity of Tpa dUTPase on dUTP at 85 °C was 90,909 U/mg. For Tpa dUTPase activity, we determined an optimum pH of 8.5 and temperature of 85 °C. Magnesium ions strongly induced activity, with an optimum concentration of 0.75 mM. The half-life of the enzyme at 94 °C was about 7 h. The specific activity of the Tpa dUTPase on dUTP was about 10-20-fold higher than that of Tpa dUTPase on dCTP. Tpa dUTPase enhanced the PCR amplification efficiency of long targets when Pfu and Vent DNA polymerases were used.

Polycation-mediated enhancement of retroviral transduction efficiency depends on target cell types and pseudotyped Env proteins: implication for gene transfer into neural stem cells.

Polycations such as polybrene (PB) are routinely used for most retroviral vector-mediated gene transfer studies because they can increase the infectivity of retroviruses. However, it was not systematically determined if addition of the polycation is an essential prerequisite for all retroviral transductions. To test this, we measured the effects of the polycation on transduction efficiency using various combinations of target cells and pseudotyped viral envelope (Env) proteins. Here, we show polycations do not always increase retroviral transduction efficiency and that their enhancing effect depends on both the type of target cells and Env proteins. The findings presented here also suggest that high transduction rates can be achieved in primary neural stem cells in vitro and in vivo by choosing an appropriate Env protein for pseudotyping without using polycations which are potentially toxic to primary cells and may change the intrinsic characteristics of cells.

Improved PCR performance using mutant Tpa-S DNA polymerases from the hyperthermophilic archaeon Thermococcus pacificus.

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)).

Improved thermostability and PCR efficiency of Thermococcus celericrescens DNA polymerase via site-directed mutagenesis.

The Thermococcus celericrescens (Tcel) DNA polymerase gene, which contains a 2328-bp open reading frame that encodes 775 amino acid residues, was expressed in the Escherichia coli strain Rosetta(DE3)pLysS. The expressed enzyme was purified through heat treatment, HisTrap™ HP column chromatography and then HiTrap™ SP HP column chromatography. Tcel DNA polymerase has poor thermostability and PCR efficiency compared to those of other family B DNA polymerases. To improve thermostability and PCR efficiency, mutant Tcel DNA polymerases were created via site-directed mutagenesis. Specifically, we targeted the A752 residue for enhanced thermostability and the N213 residue for improved PCR efficiency. The mutant Tcel DNA polymerases all showed enhanced PCR efficiency and thermostability compared to those of the wild-type Tcel DNA polymerase. Specifically, the double mutant TcelA752K/N213D DNA polymerase had an approximately three-fold increase in thermostability over that of the wild-type enzyme and amplified a long 10-kb PCR product in an extension time of 2min. However, there was a small change in the 3'→5' exonuclease activity compared with that of the wild-type Tcel DNA polymerase, even though the mutation is in the ExoII motif. The double mutant TcelA752K/N213D DNA polymerase had a 2.6-fold lower error rate compared to that of Taq DNA polymerase. It seems that the double mutant TcelA752K/N213D DNA polymerase can be used in LA (long and accurate) PCR.

Among γ-secretase substrates Notch1 alone is sufficient to block neurogenesis but does not confer self-renewal properties to neural stem cells.

Notch signaling pathway enhances neural stem cell characters and regulates cell fate decisions during neural development. Interestingly, besides Notch, other γ-secretase substrates such as APP, LRP2, and ErbB4 have also proven to have biological functions in neural development. We designed a unique experimental setting, combining gain-of- (expression of Notch intracellular domain, NICD) and loss-of-function (γ-secretase inhibition) methods, and were able to examine the function of Notch alone by excluding the activity of other γ-secretase substrates. Here, we show that the frequency and size of neurospheres generated from embryonic neural stem cells (NSCs) significantly decreased by 62.7% and 37.2%, respectively, in the presence of γ-secretase inhibitor even when NICD was expressed. Under the condition of differentiation, however, the γ-secretase inhibitor treatment did not influence the promotion of astrogenesis at the expense of neurogenesis by NICD. These results indicate that other γ-secretase substrate(s) along with Notch are important in the maintenance of the stemness of NSCs, but that Notch alone can sufficiently inhibit neurogenesis without the action of the other γ-secretase substrates during differentiation.

Cloning, expression, and PCR application of DNA polymerase from the hyperthermophilic archaeon, Thermococcus celer.

The family B DNA polymerase gene was amplified from Thermococcus celer genomic DNA by using the degenerate primers and DNA walking PCR. The Tce DNA polymerase gene was cloned and sequenced. The gene contains an ORF of 2,325 bp encoding 774 amino acid residues with a calculated molecular weight of 89,788.9 kDa. The Tce DNA polymerase was purified by heat treatment and heparin column chromatography. The optimal conditions for PCR were determined. Long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tce DNA polymerases (Tce plus DNA polymerase). Tce plus DNA polymerase surpassed the PCR performance of Tce, Taq and Pfu DNA polymerases in terms of yield and efficiency.

Purification, crystallization and preliminary crystallographic analysis of a multiple cofactor-dependent DNA ligase from Sulfophobococcus zilligii.

A recombinant DNA ligase from Sulfophobococcus zilligii that shows multiple cofactor specificity (ATP, ADP and GTP) was expressed in Escherichia coli and purified under reducing conditions. Crystals were obtained by the microbatch crystallization method at 295 K in a drop containing 1 µl protein solution (10 mg ml(-1)) and an equal volume of mother liquor [0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 10 000]. A data set was collected to 2.9 Šresolution using synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a=63.7, b=77.1, c=77.8 Å, α=83.4, ß=82.4, γ=74.6°. Assuming the presence of two molecules in the unit cell, the solvent content was estimated to be about 53.4%.

An amino acid residue in the middle of the fingers subdomain is involved in Neq DNA polymerase processivity: enhanced processivity of engineered Neq DNA polymerase and its PCR application.

Neq DNA polymerase is the first archaeal family B DNA polymerase reported to lack uracil recognition function and successfully utilize deaminated bases. We have focused on two amino acid residues (Y515, A523) in the fingers subdomain of Neq DNA polymerase, which were predicted to be located in the middle of the fingers subdomain, based on amino acid sequence alignment of the Neq DNA polymerase with structurally determined archaeal DNA polymerases. Those two residues were replaced by site-directed mutagenesis, and the enzymatic properties of the mutants were analyzed. Here, we show that the A523 residue located in the middle of the fingers subdomain affects the processivity of Neq DNA polymerase. Mutational analysis has allowed us to enhance the protein function as well as understand the function of the residues. One mutant protein, Neq A523R DNA polymerase, exhibited a roughly 3-fold enhanced processivity and extension rate compared to wild type, enabling more efficient PCR. In the presence of uracil, Neq A523R DNA polymerase outperformed Taq DNA polymerase with enhanced specificity and sensitivity. These results suggest that Neq A523R DNA polymerase could be most effectively utilized in real-time PCR using uracil-DNA glycosylase without the risk of carry-over contamination.

Molecular and biochemical characterizations of a novel arthropod endo-beta-1,3-glucanase from the Antarctic springtail, Cryptopygus antarcticus, horizontally acquired from bacteria.

Collembolan species have been known to have beta-1,3-glucanase activity and yet the genes coding such enzymes have not been demonstrated. We report here a novel arthropod endo-beta-1,3-glucanase gene CaLam from the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consists of 813bp encoding 270 amino acids with a putative signal peptide and a typical motif of glycosyl hydrolase family 16 (GHF16), E-I-D-I-T-E. The recombinant protein expressed in E. coli shows the hydrolytic activity toward laminarin (K(m) approximately 9.98mg/mL) with an optimal temperature 50 degrees C and an optimal pH 6.0. CaLam digests laminarin and laminarioligosaccharides except laminaribiose as an endo-beta-1,3-glucanase, releasing glucose, laminaribiose and laminaritriose as the major products. Analyses of molecular phylogeny of CaLam and its protein structure reveal that CaLam is closely related with bacterial beta-1,3-glucanases more than with the eukaryotic homologues. Even so, the genomic structure of the CaLam gene consisting of six exons interspersed with approximately 57 to 63bp introns confirms that it is endogenous in the genome of the Antarctic springtail. These results suggest that CaLam should have been transferred from bacteria to the lineage of the Collembolan species by horizontal gene transfer.

Biochemical properties and PCR performance of a family B DNA polymerase from hyperthermophilic Euryarchaeon Thermococcus peptonophilus.

The Thermococcus peptonophilus (Tpe) DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL in order to fully elucidate its biochemical properties and evaluate its feasibility in polymerase chain reaction (PCR) application. The expressed enzyme was then purified by heat treatment followed by two steps of column chromatography after which optimum pH and temperature of the enzyme were evaluated to be 7.0 and 75 degrees C, respectively. The optimal buffer for PCR with Tpe DNA polymerase consisted of 50 mM Tris-HCl (pH 8.0), 2 mM MgCl(2), 80 mM KCl, and 0.02% Triton X-100. Tpe DNA polymerase revealed a 3.6-fold higher fidelity (3.37 x 10(-6)) than Taq DNA polymerase (12.13 x 10(-6)) and performed significantly more efficiently in PCR amplification than both Taq and Pfu DNA polymerases. Ratios of 31:1 of Taq to Tpe DNA polymerases allowed PCR amplification of targets up to 15 kb in length with a 2.2-fold higher fidelity than Taq DNA polymerase. The results of the PCR experiments indicate that Tpe DNA polymerase may provide a higher fidelity DNA amplification in a shorter reaction time.

Production of recombinant single chain antibodies (scFv) in vegetatively reproductive Kalanchoe pinnata by in planta transformation.

We developed an asexual reproductive plant, Kalanchoe pinnata, as a new bioreactor for plant-based molecular farming using a newly developed transformation method. Leaf crenate margins were pin-pricked to infect the plant with the Agrobacterium strain LBA4404 and vacuum infiltration was also applied to introduce the target gene into the plants. Subsequently, the young mother leaf produced new clones at the leaf crenate margins without the need for time- and labor-consuming tissue culture procedures. The average transformation rates were approximately 77 and 84% for pin-prickling and vacuum-infiltration methods, respectively. To functionally characterize an introduced target protein, a nucleic acid hydrolyzing recombinant 3D8 scFv was selected and the plant based 3D8 scFv proteins were purified and analyzed. Based on abzyme analysis, the purified protein expressed with this system had catalytic activity and exhibited all of properties of the protein produced in an E. coli system. This result suggested that vegetatively reproductive K. pinnata can be a novel and potent bioreactor for bio-pharmaceutical proteins.