30 results about "Pyrococcus furiosus" patented technology

Polymerases from the Pol I family which are able to efficiently use ddNTPs have demonstrated a much improved performance when used to sequence DNA. A number of mutations have been made to the gene coding for the Pol II family DNA polymerase from the archaeon Pyrococcus furiosus with the aim of improving ddNTP utilisation. "Rational" alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural information) did not yield the desired level of selectivity for ddNTPs. However, alteration at four positions (Q472, A486, L490 and Y497) gave rise to variants which incorporated ddNTPs better than the wild type, allowing sequencing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild type Pfu-Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490Y), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y representing a 150-fold improvement over the wild type. A486, L490 and Y407 are on an alpha-helix that lines the dNTP binding groove, but the side chains of the three amino acids point away from this groove; Q472 is in a loop that connects this alpha-helix to a second long helix. None of the four amino acids can contact the dNTP directly. Therefore, the increased selectivity for ddNTPs is likely to arise from two factors: 1) Small overall changes in conformation that subtly alter the nucleotide triphosphate binding site such that ddNTPs become favoured; 2) interference with a conformational change that may be critical both for the polymerisation step and discrimination between different nucleotide triphosphates.

Purified thermostable Pyrococcus furiosus DNA polymerase that migrates on a non-denaturing polyacrylamide gel faster than phosphorylase B and Taq polymerase and more slowly than bovine serum albumin and has an estimated molecular weight of 90,000-93,000 daltons when compared with a Taq polymerase standard assigned a molecular weight of 94,000 daltons.

This invention provides isolated polynucleotides that encode replication accessory factors. The invention also provides novel DNA replication accessory factors, which have been isolated and purified from the hyperthermophilic archaea Pyrococcus furiosus. The invention also provides various methods of enhancing a nucleic acid polymerase reaction comprising the addition of the replication accessory factors to the reaction. This invention further provides methods of synthesizing, amplifying, and mutagenizing nucleic acids of interest employing the replication accessory factors. This invention also provides kits comprising at least one of the replication accessory factors. This invention also provides kits useful for various methods that comprise at least one replication accessory factor.

An enzyme preparation having optimum endo- beta -1,4-glucanase activity at a temperature of at least 90 DEG C is obtainable from or endogeneous to a strain belonging to Archaea, e.g. an endo- beta -1,4-glucanase cloned from the species Pyrococcus furiosus, DSM 3638 and being encodable by the DNA sequence of SEQ ID NO:1.

Provided herein are methods for transforming a Pyrococcus furiosus with a polynucleotide. In one embodiment, the method includes contacting a P. furiosus with a polynucleotide under conditions suitable for uptake of the polynucleotide by the P. furiosus, and identifying transformants at a frequency of, for instance, at least 10³ transformants per microgram DNA. Also provided are isolated Pyrococcus furiosus having the characteristics of Pyrococcus furiosus COM1, and plasmids that include an origin of replication that functions in a Pyrococcus furiosus. The plasmid is stable in a recipient P. furiosus without selection for more than 100 generations and is structurally unchanged after replication in P. furiosus for more than 100 generations.

A strategy to improve protein stability by domain insertion. TEM 1 beta-lactamase (BLA) and exo-inulinase, as model target enzymes, are inserted into a hyperthermophilic maltose binding protein from Pyrococcus furiosus (PfMBP). Unlike conventional protein stabilization methods that employ mutations and recombinations, the inventive approach does not require any modification on a target protein except for its connection with a hyperthermophilic protein scaffold. For that reason, target protein substrate specificity was largely maintained, which is often modified through conventional protein stabilization methods. The insertion was achieved through gene fusion by recombinant DNA techniques

The present invention relates to vectors for transforming archaea and to transformed archaea, and in particular to shuttle vector systems for transformation of members of the genus Pyrococcus.

The invention discloses a platelet-derived growth factor (PDGF) recombinant vaccine for treating pulmonary fibrosis, wherein the PDGF epitope (aa72‑87, QVRKIEIVRKKPIFKK) is inserted into the prokaryotic prokaryotic of Pyrococcus furiosus thioredoxin (PfTrx) In the plasmid pET28‑PfTrx, express and purify the recombinant vaccine PfTrx‑PDGF ¹⁶ (72‑87), the recombinant vaccine can successfully stimulate the body to produce high-titer anti-PDGF neutralizing antibodies, and significantly reduce the lung fibrosis in mice induced by bleomycin (BLM). PfTrx‑PDGF ¹⁶ (72‑87) Recombinant vaccine immunization can significantly reduce the inflammatory response of the lung tissue in the acute stage after tracheal infusion of bleomycin, inhibit the production of collagen and the increase and deposition of cell matrix in the mouse lung; at the same time, it can also reduce the TGF in the lung tissue of the mouse ‑β1, CTGF, α‑SMA, Col1a2, Col3a1 expression levels, these characteristics are extremely beneficial to slow down the process of pulmonary fibrosis.

Purified thermostable Pyrococcus furiosus DNA polymerase that migrates on a non-denaturing polyacrylamide gel faster than phosphorylase B and Taq polymerase and more slowly than bovine serum albumin and has an estimated molecular weight of 90,000-93,000 daltons when compared with a Taq polymerase standard assigned a molecular weight of 94,000 daltons.

Recombinant, alive and metabolically active bacteria including a heterologous prokaryotic biomineralized ferritin. In particular, the inventors have shown that naturally non-magnetic Escherichia coli may be engineered to become magnetic by the expression and the biomineralization of the ferritin of Pyrococcus furiosus. Moreover, the inventors have shown that a fixed number of magnetic E. coli strains keep their magnetic properties through cell division by asymmetrical division. The inventors have also shown that magnetic bacteria according to the invention may be of use in both non-therapeutic and therapeutic uses, such as, e.g., the biosensing of target substance, the depollution of complex environments, the display of antibodies, nanobodies and antigens, the delivery of therapeutic substance to target cells, the targeting and infection of target cells.

The present invention pertains to hybrid RNase H2 proteins that include fragments of amino acid sequences from Pyrococcus abyssi (P.a.), Thermococcus kodakarensis (T.kod), and Pyrococcus furiosus organisms, as well as methods of using the same to improve mismatch discrimination and activity in a high-fidelity DNA polymerase buffer.

The invention provides a probiotic honey grapefruit tea and a preparation method thereof. In the technical scheme, lactobacillus casei and streptococcus faecium are taken as probiotic components and are blended with a conventional beverage, so that an intestinal tract probiotic effect is achieved. On such basis, the invention researches an approach for inhibiting growth of two probiotics, in orderto prevent the lactobacillus casei and streptococcus faecium from propagating in the beverage and influencing the flavor of the beverage. An experiment proves that an antagonism effect on propagationefficiency can be achieved when two probiotics are co-cultured with pyrococcus furiosus and methanosarcina barkeri; on such basis, an intergrowth environment and a nutritional condition of four microorganisms are researched; a treating method according to the invention can guarantee the biomass accumulation in early stage and can achieve the balance of flora scale of four microorganisms after mixed cultivation; the blending of the beverage and the mixed bacteria acquired according to the method is capable of keeping bacteria propagation basically stagnated under storage and transportation environments, so that the flavor of the beverage is prevented from being influenced.

Provided are a method for detecting a nucleic acid based on prokaryotic Argonaute protein and an application thereof. In particular, provided is a system for detecting a target nucleic acid molecule. The system comprises guide ssDNA, a gene-editing enzyme Pyrococcus furiosus Argonaute (PfAgo), and a fluorescent reporter nucleic acid.

The invention belongs to the field of biological engineering, and in particular relates to mutational alpha-amylase as well as a preparation method and application thereof. The mutational alpha-amylase provided by the invention has a high-temperature-resistant amino acid sequence, amino acids at the following one or more sites of the amino acid sequence are replaced with other amino acids on the basis of an amino acid sequence of wild type pyrococcus furiosus alpha-amylase (PFAMY), the replaced amino acid sites are W65, C204 and N366, the expression quantity of mutated PFAMY genes reaches 10000u/l in escherichia coli, and the enzyme activity of the mutational alpha-amylase is greatly improved compared with the wild type PFAMY; the heat stability of the mutated PFAMY is improved, and the optimum temperature is changed to 100 DEG C from the original 90 DEG C, so that the mutational alpha-amylase is higher in heat stability and more stable in enzyme activity; therefore, the mutational alpha-amylase has better industrial application prospect.