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 k_cat value of the Twa H633R mutant was similar to that of wild-type, but the K_m 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.     

Molecular cloning of the gene,expression in E. coli and purification of the thermus aquaticus DNA polymerase I

The Thermus aquaticus DNA Polymerase I gene was isolated by using standard recombinant DNA techniques and cloned into an E. coli vector in front of the lac promoter. E. coli cultures produced thermostable polymerase when induced with the analog IPTG. A purification procedure is shown which renders an enzymatic preparation suitable for PCR reactions.     

几种耐热DNA聚合酶的比较

耐热DNA聚合酶广泛应用在PCR技术。本文将常用的几种耐热DNA聚合酶分为三大类,对它们的性质、差异、用途做一个简单的介绍。     

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 90 kDa consistent with the 90,470 Da 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.     

Characterization of a novel thermostable carboxylesterase from thermoalkaliphilic bacterium Bacillus thermocloaceae

A novel thermostable carboxylesterase (Est5250) of thermoalkaliphilic bacterium Bacillus thermocloaceae was heterologously expressed in Escherichia coli and its biochemical properties were investigated. Est5250 showed optimum esterase activity at 60 °C and pH 8.0. The enzyme was highly thermostable at 60 °C, interestingly, the thermostability was enhanced in the presence of Ca²⁺, retaining more than 60% of its original activity after 12 h of pre-incubation. Est5250 was active in the presence of 1% (v/v) of organic solvents and 0.1% (v/v) of non-ionic detergents. The enzyme activity was significantly enhanced up to 167% and 159% in the presence of 2-mercaptoethanol and dithiothreitol, respectively. Est5250 showed high substrate specificity for short-chain p-nitrophenyl-esters. Kinetic constants, K_m and k_cat, for p-nitrophenyl-acetate were 185.8 μM and 186.6 s^?1, respectively. Est5250 showed outstanding thermostability and tolerance to various organic solvents under thermoalkaliphilic conditions, suggesting that it would be a highly suitable biocatalyst for various biotechnological applications.

Abbreviations: B. thermocloaceae sp.: Bacillus thermocloaceae; E. coli: Escherichia coli; NP: nitrophenyl; DMSO: dimethyl sulfoxide; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMF: dimethyl formamide; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; CTAB: cetrimonium bromide; PMSF: phenylmethylsulfonyl fluoride; DEPC: diethyl pyrocarbonate; 2-ME: 2-mercaptoethanol; DTT: dithiothreitol     

同步光源之应用于病毒学研究

即将在2009年投入使用的上海同步光源（Shanghai synchrotron research facility,SSRF）属第三代设施。它的成功建设和即将投入使用给国内不同领域包括来自于物理学、材料科学和生命科学的研究人员带来前所未有的机遇。病毒学是生命科学的一个重要分支,其研究在SARS冠状病毒和禽流感爆发后越来越受到重视。本文给出了同步光源在病毒学研究中应用的一些想法。目的在于抛砖引玉,促进讨论和交流,为更好地利用光源做出努力。     

Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates.

The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 A resolution. The dNTPs bind adjacent to the O helix of Klentaq1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaq1 structures demonstrate appropriate stacking of the nucleotide base with the 3' end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant.     

Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap™ Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris–HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH₄)₂SO_4, 2 mM MgCl_2, 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).     

Characterization and PCR optimization of the thermostable family B DNA polymerase from Thermococcus guaymasensis

The gene encoding Thermococcus guaymasensis DNA polymerase (Tgu DNA polymerase) was cloned and sequenced. The 2328 bp Tgu DNA polymerase gene encoded a 775 amino acid residue protein. Alignment of the entire amino acid sequence revealed a high degree of sequence homology between Tgu DNA polymerase and other archaeal family B DNA polymerases. The Tgu DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was then purified by heat treatment followed by two steps of chromatography. The optimum pH and temperature were 7.5 and 80 °C, respectively. The optimal buffer for PCR with Tgu DNA polymerase consisted of 50 mM Tris–HCl (pH 8.2), 4 mM MgCl₂, 50 mM KCl, and 0.02% Triton X-100. Tgu DNA polymerase revealed 4-fold higher fidelity (3.17 × 10^?6) than Taq DNA polymerase (12.13 × 10^?6) and a faster amplification rate than Taq and Pfu DNA polymerases. Tgu DNA polymerase had an extension rate of 30 bases/s and a processivity of 150 nucleotides (nt). Thus, Tgu DNA polymerase has some faster elongation rate and a higher processivity than Pfu DNA polymerase. Use of different ratios of Taq and Tgu DNA polymerases determined that a ratio of 4:1 efficiently facilitated long PCR (approximately 15 kb) and a 3-fold lower error rate (4.44 × 10^?6) than Taq DNA polymerase.     

Inhibition of Taq DNA polymerase by seaweed extracts from British Columbia, Canada and Korea

Fifty-nine species of marine macrophytes from the coasts of British Columbia, Canada and Korea have been screened for the presence of PCR inhibitors, namely inhibitors of Taq DNA polymerase. Eleven of the species displayed some inhibitor activity. At the concentration of 5 μg of methanol extract in 25μL reaction mixture of PCR containing 1.5 unit of Taq DNA polymerase, one (Ulva sp.) of 8 Chlorophyta, eight (Colpomenia bullosa, Ecklonia cava, Endarachne binghamiae, Fucus distichus, Hizikia fusiformis, Sargassum confusum, Sargassum sagamianum, and Sargassum thunbergii) of 28 Phaeophyta, and one (Symphyocladia latiuscula) of 34 Rhodophyta showed inhibition in PCR amplification. In the case of the water extract, two (Cladophora columbiana, Ulva sp.) Chlorophyta, seven (Endarachne binghamiae, Fucus distichus, Hizikia fusiformis, Sargassum confusum, Sargassum sagamianum, Sargassum horneri, Scytosiphon dotyi) Phaeophyta, no Rhodophyta and one (Phyllospadix scouleri) seagrass showed inhibition in PCR amplification. the methanol fraction of Sargassum confusum and the water fraction of Fucus gardneri (mid–intertidal) have been found to inhibit PCR at level as low as 0.5 μg in 25μL of PCR reaction mixture. This revised version was published online in August 2006 with corrections to the Cover Date.     

利用PCR方法从人染色体DNA中扩增G-CSF基因

利用PCR技术从染色体基因组DNA中扩增大DNA片段具有相当大的难度。本试验采用碱变性模板以及热启动等方法,成功地扩增出1.5kb的人基因组DNA,并讨论了影响扩增大DNA片段特异性和产量的因素。     

Structure prediction and functional analyses of a thermostable lipase obtained from Shewanella putrefaciens

Previous experimental studies on thermostable lipase from Shewanella putrefaciens suggested the maximum activity at higher temperatures, but with little information on its conformational profile. In this study, the three-dimensional structure of lipase was predicted and a 60 ns molecular dynamics (MD) simulation was carried out at temperatures ranging from 300 to 400 K to better understand its thermostable nature at the molecular level. MD simulations were performed in order to predict the optimal activity of thermostable lipase. The results suggested strong conformational temperature dependence. The thermostable lipase maintained its bio-active conformation at 350 K during the 60 ns MD simulations.     

Characterization of the 5'' to 3'' exonuclease associated with Thermus aquaticus DNA polymerase.

Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.     

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.     

Characterization and structural modeling of a novel thermostable glycine oxidase from Geobacillus kaustophilus HTA426

Glycine oxidase from Geobacillus kaustophilus HTA426 (GOXK) is a 43 kDa monomer flavoenzyme containing noncovalently bound FAD. The induction of the enzyme resulted in the expression of a fully soluble protein with higher specific activity than those previously reported for GOX from B. subtilis (GOXB). A study of the kinetic properties of this novel GOXK revealed the lowest KM values for most of the substrates analyzed, with the exception of D-proline which kept a similar value and had the highest Vmax value reported. The Vmax/KM ratio maintained a substrate preference of GOXK for amines of small size, like glycine, sarcosine, N-ethyl-glycine, and glycine-ethyl-ester. GOXK presented good stability at 60-70 degrees C and in alkaline media (pH 6-9.5). The putative tridimensional structure was modeled by sequence alignment and by comparing the changes between GOXK and GOXB, and the residues that could be responsible for the substrate specificity as well as those essential for the catalytic activity were found. The comparison between the possible topology of GOXK with that of GOXB showed changes at the putative interactions between monomers for the building of the tetrameric oligomerization.     

Exploring the role of large conformational changes in the fidelity of DNA polymerase beta

The relationships between the conformational landscape, nucleotide insertion catalysis and fidelity of DNA polymerase beta are explored by means of computational simulations. The simulations indicate that the transition states for incorporation of right (R) and wrong (W) nucleotides reside in substantially different protein conformations. The protein conformational changes that reproduce the experimentally observed fidelity are significantly larger than the small rearrangements that usually accompany motions from the reactant state to the transition state in common enzymatic reactions. Once substrate binding has occurred, different constraints imposed on the transition states for insertion of R and W nucleotides render it highly unlikely that both transition states can occur in the same closed structure, because the predicted fidelity would then be many orders of magnitude too large. Since the conformational changes reduce the transition state energy of W incorporation drastically they decrease fidelity rather than increase it. Overall, a better agreement with experimental data is attained when the R is incorporated through a transition state in a closed conformation and W is incorporated through a transition state in one or perhaps several partially open conformations. The generation of free energy surfaces for R and W also allow us to analyze proposals about the relationship between induced fit and fidelity.     

活性污泥总DNA提取方法的研究

采用冻融＋玻璃珠、溶菌酶＋SDS和冻融＋玻璃珠＋溶菌酶＋SDS的方法提取活性污泥的微生物总DNA,并对以上三种方法进行比较,从中确定最佳的方法,以实现普通实验条件下成功提取符合PCR扩增要求的DNA。经紫外光度分析及电泳结果表明,冻融＋玻璃珠＋溶菌酶＋SDS方法所得的DNA的OD260/OD280为1．81,电泳结果显示,所提DNA片段分子量大于10kb,适于酶解和PCR扩增要求,为PCR技术应用于活性污泥的研究提供了一种简便、可靠的DNA提取方法。     

Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima

AIMS: Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima for the hydrolysis of lactose and the production of galacto-oligosaccharides. METHODS AND RESULTS: A putative beta-galactosidase gene of Thermotoga maritima was expressed in Escherichia coli as a carboxyl terminal His-tagged recombinant enzyme. The gene encoded a 1100-amino acid protein with a calculated molecular weight of 129,501. The expressed enzyme was purified by heat treatment, His-tag affinity chromatography, and gel filtration. The optimum temperatures for beta-galactosidase activity were 85 and 80 degrees C with oNPG and lactose, respectively. The optimum pH value was 6.5 for both oNPG and lactose. In thermostability experiments, the enzyme followed first-order kinetics of thermal inactivation and its half-life times at 80 and 90 degrees C were 16 h and 16 min, respectively. Mn2+ was the most effective divalent cation for beta-galactosidase activity on both oNPG and lactose. The Km and Vmax values of the thermostable enzyme for oNPG at 80 degrees C were 0.33 mm and 79.6 micromol oNP min(-1) mg(-1). For lactose, the Km and Vmax values were dependent on substrate concentrations; 1.6 and 63.3 at lower concentrations up to 10 mm of lactose and 27.8 mm and 139 micromol glucose min(-1) mg(-1) at higher concentrations, respectively. The enzyme displayed non-Michaelis-Menten reaction kinetics with substrate activation, which was explained by simultaneous reactions of hydrolysis and transgalactosylation. CONCLUSIONS: The results suggest that the thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galacto-oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of hydrolysis and transgalactosylation performed by beta-galactosidase of hyperthermophilic bacteria.     

Characterization of crystals of Penicillium purpurogenum acetyl xylan esterase from high-resolution X-ray diffraction

Acetyl xylan esterase from Penicillium purpurogenum, a single-chain 23 kDa member of a newly characterized family of esterases that cleaves side chain ester linkages in xylan, has been crystallized. The crystals diffract to better than 1 Å resolution at the Cornell High Energy Synchrotron Source (CHESS) and are highly stable in the synchrotron radiation. The space group is P2₁2₁2₁ and cell dimensions are a = 34.9 Å, b = 61.0 Å, c = 72.5 Å.