超嗜热古菌Thermococcus eurythermalis A501编码B家族DNA聚合酶的生化特性及PCR应用

DNA聚合酶广泛应用于PCR技术,在生命科学研究及相关领域发挥重要作用。但目前商业化DNA聚合酶仍不能完全满足科研需要,有必要寻求高性能DNA聚合酶。文中克隆表达了超嗜热古菌（Thermococcus eurythermalis）A501来源的B家族DNA聚合酶基因（NCBI数据库基因登录号为TEU＿RS04875）、表征该重组蛋白的生化特性、评价了其PCR应用。将删除intein蛋白序列的DNA聚合酶（Teu-PolB）进行体外重组表达,经亲和层析和离子交换层析纯化获得Teu-PolB蛋白;利用5′端带荧光标记的寡核苷酸作为底物,用尿素变性聚丙烯酰胺凝胶电泳鉴定Teu-PolB的生化特性;以噬菌体λDNA基因组为模板,探究Teu-PolB的PCR应用。结果显示,Teu-PolB具有DNA聚合酶活性和3′→5′核酸外切酶活性,该酶在98℃下的半衰期约为2 h,热稳定性高。使用Teu-PolB进行PCR扩增,最适PCR缓冲液为50 mmol/L Tris-HCl pH 8.0,2.5 mmol/L MgCl₂,60 mmol/L KCl,10 mmol/L （NH<...     

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 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.     

Two Family B DNA Polymerases from Aeropyrum pernix, an Aerobic Hyperthermophilic Crenarchaeote

DNA polymerase activities in fractionated cell extract of Aeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3'-->5' exonuclease and polymerase activities. The product of polA gene (Pol I) was aphidicolin resistant and heat stable up to 80 degrees C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95 degrees C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of Pyrodictium occultum. The deduced amino acid sequence of A. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain Crenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.     

Characterization of novel hexadecameric thioredoxin peroxidase from Aeropyrum pernix K1

A gene (APE2278) encoding the peroxiredoxin (Prx) homologous protein of yeast and human was identified in the genome data base of the aerobic hyperthermophilic archaeon Aeropyrum pernix. We cloned the gene and produced the encoded protein in Escherichia coli cells. The isolated recombinant protein showed peroxidase activity in vitro and used the thioredoxin system of A. pernix as an electron donor. These results indicate that the recombinant protein is in fact thioredoxin peroxidase (ApTPx) of A. pernix. Immunoblot analysis revealed that the expression of ApTPx was induced as a cellular adaptation in response to the addition of exogenous H2O2 and may exert an antioxidant activity in vivo. An analysis of the ApTPx oligomers by high pressure liquid chromatography and electron microscopic studies showed that ApTPx exhibited the hexadecameric protein forming 2-fold toroid-shaped structure with outer and inner diameters of 14 and 6 nm, respectively. These results indicated that ApTPx is a novel hexadecameric protein composed of two identical octamers. Although oligomerization of individual subunits does not take place through an intersubunit-disulfide linkage involving Cys50 and Cys213, Cys50 is essential for the formation of the hexadecamer. Mutagenesis studies suggest that the sulfhydryl group of Cys50 is the site of oxidation by peroxide and that oxidized Cys50 reacts with the sulfhydryl group of Cys213 of another subunit to form an intermolecular disulfide bond. The resulting disulfide can then be reduced by thioredoxin. In support of this hypothesis, ApTPx mutants lacking either Cys50 or Cys213 showed no TPx activity, whereas the mutant lacking Cys207 had a TPx activity. This is the first report on the biochemical and structural features of a novel hexadecameric thioredoxin peroxidase from the archaea.     

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 of a novel thermostable O-acetylserine sulfhydrylase from Aeropyrum pernix K1

An O-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon Aeropyrum pernix K1, which shares the pyridoxal 5'-phosphate binding motif with both OASS and cystathionine beta-synthase (CBS), was cloned and expressed by using Escherichia coli Rosetta(DE3). The purified protein was a dimer and contained pyridoxal 5'-phosphate. It was shown to be an enzyme with CBS activity as well as OASS activity in vitro. The enzyme retained 90% of its activity after a 6-h incubation at 100 degrees C. In the O-acetyl-L-serine sulfhydrylation reaction, it had a pH optimum of 6.7, apparent K(m) values for O-acetyl-L-serine and sulfide of 28 and below 0.2 mM, respectively, and a rate constant of 202 s(-1). In the L-cystathionine synthetic reaction, it showed a broad pH optimum in the range of 8.1 to 8.8, apparent K(m) values for L-serine and L-homocysteine of 8 and 0.51 mM, respectively, and a rate constant of 0.7 s(-1). A. pernix OASS has a high activity in the L-cysteine desulfurization reaction, which produces sulfide and S-(2,3-hydroxy-4-thiobutyl)-L-cysteine from L-cysteine and dithiothreitol.     

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.     

Crystal structure of translation initiation factor 5B from the crenarchaeon Aeropyrum pernix

Initiation factor 5B (IF5B) is a universally conserved translational GTPase that catalyzes ribosomal subunit joining. In eukaryotes, IF5B directly interacts via a groove in its domain IV with initiation factor 1A (IF1A), another universally conserved initiation factor, to accomplish efficient subunit joining. Here, we have determined the first structure of a crenarchaeal IF5B, which revealed that the archaea‐specific region of IF5B (helix α15) binds and occludes the groove of domain IV. Therefore, archaeal IF5B cannot access IF1A in the same manner as eukaryotic IF5B. This fact suggests that different relationships between IF5B and IF1A exist in archaea and eukaryotes. Proteins 2016; 84:712–717. © 2016 Wiley Periodicals, Inc.     

Characterization of native glutamate dehydrogenase from an aerobic hyperthermophilic archaeon Aeropyrum pernix K1

Glutamate dehydrogenase (GDH) was purified and characterized from an aerobic hyperthermophilic archaeon Aeropyrum pernix (A. pernix) K1. The enzyme has a hexameric structure with a native molecular mass of about 285 +/- 15 kDa. It was specific for NADP and thermostable (74% activity was remained after 5 h incubation at 100 degrees C). The activity of the enzyme increased in the presence of polar water-miscible organic solvents such as acetonitrile, methanol, and ethanol. The N-terminal sequence of GDH is Met-Gln-Pro-Thr-Asp-Pro-Leu-Glu-Glu-Ala. This sequence, except for the methionine, corresponds to amino acids 7-15 of the open reading frame (ORF) encoding the predicted GDH (ORF APE 1386). In the ORF nucleotide sequence, the codon TTG appears at the position of the methionine, suggesting that the leucine codon might be recognized as an initiation codon and translated to methionine in A. pernix GDH.     

Provirus induction in hyperthermophilic archaea: characterization of Aeropyrum pernix spindle-shaped virus 1 and Aeropyrum pernix ovoid virus 1

By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum pernix, and under special conditions of A. pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae.     

Biochemical analysis of a DNA replication origin in the archaeon Aeropyrum pernix

We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple origin recognition box (ORB) sites that we show to be a replication origin. At this origin, there are four ORB elements disposed either side of an A+T-rich region. An ORC1 protein dimer binds at each of these ORB sites. Once all four ORB sites have bound ORC1 protein, there is a transition to a higher-order assembly with a defined alteration in topology and superhelicity. Furthermore, after this transition, the A+T-rich region becomes sensitive to digestion by DNase I and P1 nuclease, revealing that the transition promotes distortion of the DNA in this region, presumably as a prelude to loading of MCM helicase.     

Aeropyrum pernix K1磷脂酶A2的克隆、表达及其性质

从超嗜热需氧古细菌AeropyrumpernixK1中抽提出染色体基因组,经PCR扩增得到磷脂酶A2基因,用带有His-tag标记的pET15b作为表达载体,在大肠杆菌BLP中成功地诱导表达。表达产物经过Ni-螯合柱一步得到纯化。SDS-PAGE检测只有一条带,其准确分子量为17,871kD。对纯化后的磷脂酶A2测定其酶活性和生物活性,得出其最适反应温度为90℃,最适pH范围为7·8~8·2。至此首次成功地在大肠杆菌中表达了古细菌嗜热磷脂酶A2,这将为以后对该酶的结构和功能以及耐热机制研究打下很好的基础,同时有利于古细菌研究领域的扩展。     

Biochemical characterization of DNA-binding proteins from Pyrobaculum aerophilum and Aeropyrum pernix

Several representatives of the Crenarchaeal branch of the Archaea contain highly abundant, small, positively charged proteins exemplified by the Sso7d protein from Sulfolobus solfataricus. These proteins bind to DNA in a non-sequence-specific manner. Using publicly available genomic sequence information, we identified a second class of small Crenarchaeal DNA-binding proteins represented by the Pyrobaculum aerophilum open reading frame 3192–encoded (Pae3192) protein and its paralogs. We investigated the biochemical properties of the Pae3192 protein and an orthologous protein (Ape1322b) from Aeropyrum pernix in side-by-side experiments with the Sso7d protein. We demonstrate that the recombinant Ape1322b, Pae3192 and Sso7d proteins bind to DNA and that the DNA-protein complexes formed are slightly different for each protein. We show that like Sso7d, Pae3192 constrains negative supercoils in DNA. In addition, we show that all three proteins raise the melting temperature of duplex DNA upon binding. Finally, we present the equilibrium affinity constants and kinetic association constants of each protein for single-stranded and double-stranded DNA.     

Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix

Archaea contain one or more proteins with homology to eukaryotic ORC/Cdc6 proteins. Sequence analysis suggests the existence of at least two subfamilies of these proteins, for which we propose the nomenclature ORC1 and ORC2. We have determined crystal structures of the ORC2 protein from the archaeon Aeropyrum pernix in complexes with ADP or a non-hydrolysable ATP analogue, ADPNP. Between two crystal forms, there are three crystallographically independent views of the ADP complex and two of the ADPNP complex. The protein molecules in the three complexes with ADP adopt very different conformations, while the two complexes with ADPNP are the same. These structures indicate that there is considerable conformational flexibility in ORC2 but that ATP binding stabilises a single conformation. We show that the ORC2 protein can bind DNA, and that this activity is associated with the C-terminal domain of the protein. We present a model for the interaction of the winged helix (WH) domain of ORC2 with DNA that differs from that proposed previously for Pyrobaculum aerophilum ORC/Cdc6.     

Crystal structure of archaeal chromatin protein Alba2-double-stranded DNA complex from Aeropyrum pernix K1

All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.     

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 μM, a K(M) for phosphoenolpyruvate of 891 μM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.     

Cloning, expression and characterisation of a Family B ATP-dependent phosphofructokinase activity from the hyperthermophilic crenarachaeon Aeropyrum pernix

Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase.     

A novel member of the protein disulfide oxidoreductase family from Aeropyrum pernix K1: structure, function and electrostatics

The formation of disulfide bonds between cysteine residues is a rate-limiting step in protein folding. To control this oxidative process, different organisms have developed different systems. In bacteria, disulfide bond formation is assisted by the Dsb protein family; in eukarya, disulfide bond formation and rearrangement are catalyzed by PDI. In thermophilic organisms, a potential key role in disulfide bond formation has recently been ascribed to a new cytosolic Protein Disulphide Oxidoreductase family whose members have a molecular mass of about 26 kDa and are characterized by two thioredoxin folds comprising a CXXC active site motif each. Here we report on the functional and structural characterization of ApPDO, a new member of this family, which was isolated from the archaeon Aeropyrum pernix K1. Functional studies have revealed that ApPDO can catalyze the reduction, oxidation and isomerization of disulfide bridges. Structural studies have shown that this protein has two CXXC active sites with fairly similar geometrical parameters typical of a stable conformation. Finally, a theoretical calculation of the cysteine pK(a) values has suggested that the two active sites have similar functional properties and each of them can impart activity to the enzyme. Our results are evidence of functional similarity between the members of the Protein Disulphide Oxidoreductase family and the eukaryotic enzyme PDI. However, as the different three-dimensional features of these two biological systems strongly suggest significantly different mechanisms of action, further experimental studies will be needed to make clear how different three-dimensional structures can result in systems with similar functional behavior.