Cloning,Sequence Analysis and Three-dimensional Structure Prediction of DNA Pol I from Thermophilic <Emphasis Type="Italic">Geobacillus</Emphasis> sp. <Emphasis Type="Italic">MKK</Emphasis> Isolated from an Iranian Hot Spring

Molecular phylogenetic analysis of a novel thermophilic eubacterium isolated from an Iranian hot spring using 16S rDNA sequence showed that the new isolate belongs to genera Geobacillus. DNA pol I gene from this isolate was amplified, cloned, sequenced, and the three-dimensional (3D) structure of deduced amino acid sequence was predicted. Sequence analysis revealed the gene is 2,631 bp long, encodes a protein of 876 amino acids with a calculated molecular mass of 99 kDa, and belongs to family A DNA polymerases. Comparison of 3′–5′exonuclease domain of Klenow fragment (KF) with corresponding region of newly identified DNA pol I (MF), the large fragment of Bacillus stearothermophilus DNA pol I (BF) and Klentaq1, revealed not only deletions in three regions compared to KF, but that three of the four critical metal-binding residues in KF (Asp355, Glu357, Asp424, and Asp501) are altered in MF as well. Predicted 3D structure and sequence alignments between MF and BF showed that all critical residues in the polymerase active site are conserved.     

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.     

Molecular Characterization of Mosquitocidal Toxin (Surface Layer Protein,SLP) from <Emphasis Type="Italic">Bacillus cereus</Emphasis> VCRC B540

A marine Bacillus cereus (VCRC B540) with mosquitocidal effect was recently reported from red snapper fish (Lutjanus sanguineous) gut and surface layer protein (S-layer protein, SLP) was reported to be mosquito larvicidal factor. In this present study, the gene encoding the surface layer protein was amplified from the genomic DNA and functionally characterized. Amplification of SLP-encoding gene revealed 1,518 bp PCR product, and analysis of the sequence revealed the presence of 1482 bp open reading frame with coding capacity for a polypeptide of 493 amino acids. Phylogenetic analysis revealed with homology among closely related Bacillus cereus groups of organisms as well as Bacillus strains. Removal of nucleotides encoding signaling peptide revealed the functional cloning fragment of length 1398 bp. Theoretical molecular weight (51.7 kDa) and isoelectric point (5.99) of the deduced functional SLP protein were predicted using ProtParam. The amplified PCR product was cloned into a plasmid vector (pGEM-T), and the open reading frame free off signaling peptide was subsequently cloned inpET-28a(+) and expressed in Escherichia coli BL21 (DE3). The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant SLP was confirmed using western blotting, and functional SLP revealed mosquito larvicidal property. Therefore, the major findings revealed that SLP is a factor responsible for mosquitocidal activity, and the molecular characterization of this toxin was extensively studied.     

Replacing 32 proline residues by a noncanonical amino acid results in a highly active DNA polymerase

Protein engineering may be achieved by rational design, directed evolution-based methods, or computational protein design. Mostly these methods make recourse to the restricted pool of the 20 natural amino acids. With the ability to introduce different new kinds of functionalities into proteins, the use of noncanonical amino acids became a promising new method in protein engineering. Here, we report on the generation of a multifluorinated DNA polymerase. DNA polymerases are highly dynamic enzymes that catalyze DNA synthesis in a template-dependent manner, thereby passing several conformational states during the catalytic cycle. Here, we globally replaced 32 proline residues by the noncanonical imino acid (4R)-fluoroproline in a DNA polymerase of 540 amino acids (KlenTaq DNA polymerase). Interestingly, the substitution level of the proline residues was very efficient (92%). Nonetheless, the introduction of (4R)-fluoroproline into the DNA polymerase resulted in a highly active fluorinated enzyme, which was investigated in primer extension and PCR assays to analyze activity, selectivity, and stability in comparison to the parental enzyme. The DNA polymerase retained fidelity, activity, and sensitivity as the parental wild-type enzyme accompanied by some loss in thermostability. These results demonstrate that a noncanonical amino acid can be used for substitutions of natural counterparts in a highly dynamic enzyme with high molecular weight without effecting crucial enzyme properties. Furthermore, the employed DNA polymerase represents a promising starting point for directed DNA polymerase evolution with noncanonical amino acids.     

Gene Cloning,Overexpression, and Characterization of the Nitrilase from <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> tg1-A6 in <Emphasis Type="Italic">E. coli</Emphasis>

A DNA fragment containing the entire coding sequence of nitrilase gene was amplified from Rhodococcus rhodochrous tg1-A6 with high nitrilase activity using PCR and sequenced. The open reading frame of the nitrilase gene contains 1,101 base pairs, which encodes a putative polypeptide of 366 amino acid residues. The nitrilase gene was cloned into an expression vector pET-28a and expressed in an Escherichia coli strain BL21(DE3). The enzymatic activity of nitrilase, which converts various nitriles to the corresponding carboxylic acids, was detected to reach 24.5 U/ml at 9 h in the recombinant bacteria.     

Cloning and expression of L-asparaginase gene in Escherichia coli

The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved regions of published ASN gene sequences. Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed in different E. coli host strains. The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain. The recombinant plasmid in E. coli AS1.357 remained stable after 72h of cultivation and 5h of heat induction without selective pressure. The ASN gene of E. coli AS1.357 was sequenced and had high homology compared to the reported data.     

Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from <Emphasis Type="Italic">Pseudomonas rhodesiae</Emphasis> CIP 107491 and Production of the Recombinant Protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The alpha-pinene oxide lyase (Prα-POL) from Pseudomonas rhodesiae CIP107491 belongs to catabolic alpha-pinene degradation pathway. In this study, the gene encoding Prα-POL has been identified using mapping approach combined to inverse PCR (iPCR) strategy. The Prα-POL gene included a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7 kDa protein, with a theoretical isoelectric point (pI) of 5.23. The amino acids sequence analysis showed homologies with those of proteins with unknown function from GammaProteobacteria group. Identification of a conserved domain in amino acid in positions 18 to 190 permitted to classify Prα-POL among the nuclear transport factor 2 (NTF2) protein superfamily. Heterologous expression of Prα-POL, both under its native form and with a histidin tag, was successfully performed in Escherichia coli, and enzymatic kinetics were analyzed. Bioconversion assay using recombinant E. coli strain allowed to reach a rate of isonovalal production per gramme of biomass about 40-fold higher than the rate obtained with P. rhodesiae.     

Synthesis of photolabile dUTP analogues and their enzymatic incorporation for DNA labeling

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Evidence Showing Duplication and Recombination of cel Genes in Tandem from Hyperthermophilic Thermotoga sp.

This study was conducted to assess the gene duplication and diversification of tandem cellulase genes in thermophilic bacteria. The tandem cellulase genes cel5C and cel5D were cloned from Thermotoga maritima MSB8, and a survey of the thermophilic bacterial genome for tandem cel genes from the databases was carried out. A clone having 2.3?kb fragment from T. maritima MSB8 showed cellulase activity, which had two open reading frames in tandem (cel5C and cel5D). The cel5C gene has 954?bp, which encodes a protein of 317 amino acid residues with a signal peptide of 23 amino acids, and the other gene cel5D consisting of 990?bp encoding a protein of 329 amino acid residues. These two proteins have similarity with the enzymes of glycosyl hydrolase family 5. From the enzyme assay, it was observed that Cel5C was extracellular and Cel5D was intracellular cellulase. Phylogenetic and homology matrix analyses of DNA and protein sequences revealed that family 12 cellulase enzymes Cel12A and Cel12B displayed higher homology (>50?%), but Cel5C and Cel5D enzymes belong to family 5 displayed lower homology (<30?%). In addition, repeated and mirror sequences in tandem genes are supposed to show the existence of gene duplication and recombination.     

White spot syndrome virus Orf514 encodes a bona fide DNA polymerase

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome, one of the most devastating diseases in shrimp aquaculture. The genome of WSSV includes a gene that encodes a putative family B DNA polymerase (ORF514), which is 16% identical in amino acid sequence to the Herpes virus 1 DNA polymerase. The aim of this work was to demonstrate the activity of the WSSV ORF514-encoded protein as a DNA polymerase and hence a putative antiviral target. A 3.5 kbp fragment encoding the conserved polymerase and exonuclease domains of ORF514 was overexpressed in bacteria. The recombinant protein showed polymerase activity but with very low level of processivity. Molecular modeling of the catalytic protein core encoded in ORF514 revealed a canonical polymerase fold. Amino acid sequence alignments of ORF514 indicate the presence of a putative PIP box, suggesting that the encoded putative DNA polymerase may use a host processivity factor for optimal activity. We postulate that WSSV ORF514 encodes a bona fide DNA polymerase that requires accessory proteins for activity and maybe target for drugs or compounds that inhibit viral DNA replication.     

Biochemical Properties and PCR Performance of a Family B DNA Polymerase from Hyperthermophilic Euryarchaeon <Emphasis Type="Italic">Thermococcus peptonophilus</Emphasis>

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 °C, respectively. The optimal buffer for PCR with Tpe DNA polymerase consisted of 50 mM Tris–HCl (pH 8.0), 2 mM MgCl₂, 80 mM KCl, and 0.02% Triton X-100. Tpe DNA polymerase revealed a 3.6-fold higher fidelity (3.37 × 10⁻⁶) than Taq DNA polymerase (12.13 × 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.     

Cloning and Sequence Analysis of Novel DNA Polymerases from Thermophilic Geobacillus Species Isolated from Hot Springs in Turkey: Characterization of a DNA Polymerase I from Geobacillus kaue Strain NB

The complete coding sequences of the polA genes from seven thermophilic Geobacillus species, isolated from hot springs of G?nen and Hisaralan in Turkey, were cloned and sequenced. The polA genes of these Geobacillus species contain a long open reading frame of 2,637 bp encoding DNA polymerase I with a calculated molecular mass of 99 kDa. Amino acid sequences of these Geobacillus DNA polymerases are closely related. The multiple sequence alignments show all include the conserved amino acids in the polymerase and 5'-3' exonuclease domains, but the catalytic residues varied in 3'-5' exonuclease domain of these Geobacillus DNA polymerases. One of them, DNA polymerase I from Geobacillus kaue strain NB (Gkaue polI) is purified to homogeneity and biochemically characterized in vitro. The optimum temperature for enzymatic activity of Gkaue polI is 70 °C at pH 7.5-8.5 in the presence of 8 mM Mg(2+) and 80-100 mM of monovalent ions. The addition of polyamines stimulates the polymerization activity of the enzyme. Three-dimensional structure of Gkaue polI predicted using homology modeling confirmed the conservation of all the functionally important regions in the polymerase active site.     

A New Aminopeptidase from the Keratin-Degrading Strain Streptomyces fradiae var. k11

An aminopeptidase gene fragment was isolated from a keratin-degrading strain, Streptomyces fradiae var. k11, by PCR amplification using a degenerate primer set designed based on the partial amino acid sequence of the native enzyme. The gene, designated sfap, encoded a polypeptide of 461 amino acids comprised of three domains: a signal peptide, a mature region, and a C-terminal propeptide. The aminopeptidase, SFAP, had highest amino acid sequence identity (79%) with a putative aminopeptidase from Streptomyces griseus subsp. griseus NBRC 13350. The gene with and without C-terminal propeptide was successfully overexpressed in Escherichia coli BL21 (DE3), and the gene without C-terminal propeptide encoded a functional enzyme. Purified recombinant SFAP exhibited optimal activity at pH 8.0 and 60 °C, and retained >60% peak activity over a broad range of temperature. The enzyme was thermal and pH stable, and showed metalloprotease characteristics, which was inhibited by EDTA but activated by Ca²⁺ and Co²⁺. This is the first study to report the gene cloning and expression of a leucine aminopeptidase from S. fradiae.     

Tightening the Belt on Polymerases: Evaluating the Physical Constraints on Enzyme Substrate Size

To test the limits of polymerase enzyme activity on geometrically constrained DNAs, four very small synthetic circular DNAs were constructed by using newly developed methods. Surprisingly, even a 13-nucleotide circular DNA (1) can be copied successfully by both DNA and RNA polymerases, despite the very small diameter and large degree of distortion in this synthetic DNA. The picture shows models to indicate the relative sizes of 1 and the Klenow fragment of the DNA polymerase I from E. coli.     

Eplt4 Proteinaceous Elicitor Produced in Pichia pastoris Has a Protective Effect Against Cercosporidium sofinum Infections of Soybean Leaves

A complementary DNA library was constructed from the mycelium of Trichoderma asperellum T4, and a highly expressed gene fragment named EplT4 was found. In order to find a more efficient and cost-effective way of obtaining EplT4, this study attempted to produce EplT4 using a Pichia pastoris expression system. The gene encoding EplT4, with an additional 6-His tag at the C-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115 to obtaining more protein for the further research. Transformants of P. pastoris were selected by PCR analysis, and the ability to secrete high levels of the EplT4 protein was determined. The optimal conditions for induction were assayed using the shake flask method and an enzyme-linked immunosorbent assay. The yield of purified EplT4 was approximately 20 mg/L by nickel affinity chromatography and gel-filtration chromatography. Western blot and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis revealed that the recombinant EplT4 was expressed in both its monomers and dimers. Soybean leaves treated with the EplT4 monomer demonstrated the induction of glucanase, chitinase III-A, cysteine proteinase inhibitor, and peroxidase genes. Early cellular events in plant defense response were also observed after incubation with EplT4. Soybean leaves protected by EplT4 against the pathogen Cercosporidium sofinum (Hara) indicated that EplT4 produced in P. pastoris was biologically active and would be potentially useful for improving food security.     

Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-ΔSHP-1 Antibodies

This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1（designated ΔSHP-1） and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta（DE3） E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.     

A novel amylomaltase from Corynebacterium glutamicum and analysis of the large-ring cyclodextrin products

Amylomaltase catalyzes the formation of large-ring cyclodextrins (LR-CDs) from starch. This study aims to construct the recombinant amylomaltase from Corynebacterium glutamicum and to characterize the purified enzyme with the emphasis on the profile of LR-CDs production. A novel amylomaltase from Corynebacterium glutamicum ATCC 13032 was cloned and expressed in Escherichia coli BL21 (DE3) using the expression vector pET-19b. The open reading frame of amylomaltase gene of 2,121 bp (encoding the polypeptide of 706 amino acid residues) was obtained with the N-terminal His-tag fragment of 69 bp attached before the start codon of the amylomaltase gene. The deduced amino acid sequence showed a low sequence identity (20?C25%) to those thermostable amylomaltases from Thermus sp. The maximum enzyme activity was obtained when the recombinant cells were cultured at 37 °C for 2 h after induction with 0.4 mM isopropyl thio-??-D-galactoside (IPTG). The enzyme was 11-fold purified with a yield of 30% by a HiTrap affinity column. The purified amylomaltase showed a single band of 84 kDa on a 7.5% SDS-PAGE. When the enzyme acted on pea starch, it catalyzed an intramolecular transglucosylation (cyclization) reaction that produced LR-CDs or cycloamyloses (CA). The product profile was dependent on the incubation time and the enzyme concentration. Shorter incubation time gave larger LR-CDs as principal products. At 4 h incubation, the product was composed of a mixture of LR-CDs in the range of CD19?CCD50, with CD27?C28 as products with highest amount. It is noted that CD19 was the smallest product in all conditions tested. The enzyme also catalyzes intermolecular transglucosylation on various malto-oligosaccharides, with maltose as the smallest substrate.     

Purification and Characterization of a New Serine Protease with Fibrinolytic Activity from the Marine Invertebrate, Urechis unicinctus

A non-hemorrhagic, chymotrypsin-like serine protease, UFEII, was purified from the marine echiuroid worm, Urechis unicinctus, after a combination of chromatography steps. UFEII was monomeric, with an apparent molecular weight of 26.7 kDa via SDS-PAGE. The isoelectric point of UFEII was 4.03, and the maximum activity of the enzyme was observed at 50 °C and pH?8.0. According to fibrin plate assays, UFEII could not only directly degrade fibrin and fibrinogen but also activate plasminogen. Further, UFEII preferentially hydrolyzed the fibrinogen γ-chain, followed by the Bβ-chains and Aα-chains. Moreover, ufeII, full length of the gene encoding UFEII, was obtained by RT-PCR, degenerated PCR, and nested PCR. The ufeII was determined to be a 906-bp cDNA containing an open reading frame of 795 bp encoding a putative protein of 264 amino acids with a predicted molecular weight of 27.03 kDa. Besides, UFEII exhibited no hemorrhagic effect. Overall, U. unicinctus may represent a potential source of new therapeutic agents in thrombolytic therapy.     

Exonuclease activity of proofreading DNA polymerases is at the origin of artifacts in molecular profiling studies

CE fingerprint methods are commonly used in microbial ecology. We have previously noticed that the position and number of peaks in CE-SSCP (single-strand conformation polymorphism) profiles depend on the DNA polymerase used in PCR [1]. Here, we studied the fragments produced by Taq polymerase as well as four commercially available proofreading polymerases, using the V3 region of the Escherichia coli rss gene as a marker. PCR products rendered multiple peaks in denaturing CE; Taq polymerase was observed to produce the longest fragments. Incubation of the fragments with T4 DNA polymerase indicated that the 3'-ends of the proofreading polymerase amplicons were recessed, while the Taq amplicon was partially +A tailed. Treatment of the PCR product with proofreading DNA polymerase rendered trimmed fragments. This was due to the 3'-5' exonuclease activity of these enzymes, which is essential for proofreading. The nuclease activity was reduced by increasing the concentration of dNTP. The Platinum Pfx DNA polymerase generated very few artifacts and could produce 85% of blunted PCR products. Nevertheless, despite the higher error rate, we recommend the use of Taq polymerase rather than proofreading in the framework for molecular fingerprint studies. They are more cost-effective and therefore ideally suited for high-throughput analysis; the +A tail artifact rate can be controlled by modifying the PCR primers and the reaction conditions.     

A recombinant cyclodextrin glycosyltransferase cloned from Paenibacillus sp. strain RB01 showed improved catalytic activity in coupling reaction between cyclodextrins and disaccharides

Recombinant cyclodextrin glycosyltransferase (CGTase) was obtained by cloning the PCR gene fragment from thermotolerant Paenibacillus sp. strain RB01 screened from hot spring area in Thailand and cloned into the Escherichia coli expression vector. The nucleotide sequence was analyzed and aligned. Nucleotide sequence of the recombinant CGTase contained an open reading frame of 2139 bp encoding 713 amino acid residues. The recombinant required one-third of culture time and neutral pH to produce CGTase compared to wild type. CGTases from both wild type and transformant were purified in parallel by starch adsorption and DEAE cellulose column. Their biochemical properties such as molecular weight, optimum pH and temperature were quite similar. However, the recombinant enzyme showed improved catalytic activity in the coupling reaction between cyclodextrins (CDs) and some disaccharides. Among several sugars tested with excess βCD, cellobiose was the best substrate followed by leucrose. Very low activity was observed with trehalose, lactose and mellibiose. Sucrose and raffinose showed no activity. The K _m and other kinetic parameters of recombinant enzyme were determined for cellobiose and several cyclodextrin derivatives. Recombinant CGTase showed lower K _m for βCD and its derivatives, with improved activity compared to wild type enzyme.