41 results about "Aminoacyl-tRNA" patented technology

The present invention relates to a novel function of lysyl tRNA synthetase (KRS) which enhances tumor cell migration and affects cancer metastasis via KRS's interaction with laminin receptor (67LR) by its translocation to membrane. More particularly, the present invention relates to a method for modulating cancer metastasis or migration, which comprises regulating intracellular levels of KRS; a composition for preventing or treating cancer; use of expression vector for inhibiting the expression of KRS; a method for preventing or treating cancer; use of an agent for inhibiting an activity of KRS; a method for screening an agent which modulates cancer metastasis or migration; and a method for screening an agent which inhibits the interaction of KRS with 67LR, by said novel function. Thus, KRS can modulate cancer metastasis or migration and furthermore, can modulate intra-cellular metabolism related to 67LR. The interaction between KRS and 67LR can be used effectively in treating, preventing and / or diagnosing of various diseases or disorders related to the interaction.

Thrombopoietic compositions are provided comprising tyrosyl tRNA synthetase polypeptides, including truncations and / or variants thereof. Also provided are methods of using such compositions in the treatment of conditions that benefit from increased thrombopoiesis, such as thrombocytopenia.

Object is to provide a method of constructing a library of peptides having, at a desired position in the random sequence of each peptide, an amino acid having a portion capable of binding to a target. The invention provides a method of producing a library of peptides having, at a designated position in the random sequence of each peptide, a special amino acid having a portion capable of binding to a target, including (i) preparing a library of mRNAs having, in the mRNA sequence coding for a random amino acid sequence, a base sequence having an altered codon encoding the special amino acid, (ii) preparing an aminoacyl tRNA with the special amino acid linked to a tRNA encoded by the altered codon, and (iii) translating the mRNAs by using a cell-free translation system containing the aminoacyl tRNA to obtain a library of peptides having, in the random sequence thereof, a predetermined special amino acid.

The invention discloses viromembrane protein with site-specific mutagenesis and site-specific decoration, a preparation method and applications of the viromembrane protein. Amber codon TAG is introduced to the specific site of envelope protein G (VSV G) gene of vesicular stomatitis virus, unnatural amino acid DiZPK with photo-crosslinking property is introduced to the specific site of VSV G by utilizing the orthorhombic aminoacyl-tRNA synthetase-tRNA through site-specific mutagenesis. In the interaction process of virus and host cells, the DiZPK photo-crosslinking reaction is triggered by 365nm ultraviolet light, the covalent bond is formed between the VSV G and interacting protein, the interaction dissociation can be avoided, and the powerful measure is provided for the research of the interaction of the virus and protein of the host cells.

The invention relates to a building method of a stable cell line carrying orthogonal tRNA / arginyl tRNA synthetase. On the basis of a gene codon expansion technology, a pair of orthogonal tRNA / arginyl tRNA synthetase is used for introducing unnatural amino acid into a fixed point of protein. The invention also relates to a building method of double lentiviral vectors, a building method of a multi-copy-number tRNA carrying carrier and a method for stably integrating the orthogonal tRNA / arginyl tRNA synthetase gene into the cell genome through double lentiviral stable transduction and plasmid stable transfection. The invention further relates to application of the stable cell line, such as the purpose of expressing the target protein containingunnatural amino acid.

The invention relates to an aminoacyl-tRNA (transfer ribonucleic acid) synthetase mutant containing an amino acid sequence selected from groups consisting of amino acids shown by SEQ ID NO:4 and conservative variants thereof. The invention provides a CpK (short for N<epsilon>-(1-methylcyclopropyl-2-acrylamide)-lysine) translation system for fixed point specific insertion of N<epsilon>-(1-methylcyclopropyl-2-acrylamide)-lysine in a target protein through pairing of orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase, and a method for fixed point specific insertion of CpK in the target protein by using the translation system. The CpK translation system comprises (i) N<epsilon>-(1-methylcyclopropyl-2-acrylamide)-lysine, (ii) the orthogonal aminoacyl-tRNA synthetase, (iii) the orthogonal tRNA and (iv) a nucleic acid for coding the target protein, wherein the orthogonal aminoacyl-tRNA synthetase is used for realizing aminoacylation of the orthogonal tRNA preferentially by using CpK, and the nucleic acid contains at least one selective codon specifically identified by the orthogonal tRNA. The invention also relates to an application of a mutant protein for fixed point specific insertion of CpK in protein labeling through a light click reaction with a tetrazole compound.

The invention relates to an aminoacyl-tRNA (transfer ribonucleic acid) synthetase mutant, wherein the amino acid sequence contained therein is selected from a group composed of the amino acids shown by the SEQ ID No. 2, 3 and 4 and the conservative mutants thereof. The invention provides a 3-chlorinated tyrosine translation system doping 3-chlorinated tyrosine into the target protein by use of the orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase and the pairing thereof, and a method for doping the 3-chlorinated tyrosine into the target protein by use of the translation system. The 3-chlorinated tyrosine translation system comprises (i) 3-chlorinated tyrosine, (ii) orthogonal aminoacyl-tRNA synthetase, (iii) orthogonal tRNA and (iv) nucleic acid coding the target protein, wherein the orthogonal aminoacyl-tRNA synthetase performs aminoacylation of the orthogonal tRNA by use of the 3-chlorinated tyrosine by priority; and the nucleic acid contains at least one selection coder for specificity identification of the orthogonal tRNA.

The present invention relates to a new use for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Specifically, the present invention provides a new method for evaluating the aminoacylation state of tRNAs, which are useful for site-directed mutagenesis of both sidechain and backbone protein structures. Further, the present invention useful for the generation of novel peptides, polypeptides, and proteins that may be used in drug design and screening, and in the design of small molecule agonists or antagonists for receptors, enzymes and other proteins and molecules involved in various disease states. Additionally, the present invention is useful in the field of tRNA aminoacylation, particularly in the design of synthetases.

A method of expressing a protein having an unnatural amino acid integrated thereinto comprising expressing (A) a mutant tyrosyl tRNA synthase that is derived from Escherichia coli-origin tyrosyl tRNA synthase and has an elevated specificity for an unnatural tyrosine derivative compared with the specificity for tyrosine, (B) a suppressor tRNA originating in an eubacterium belonging to the genus Bacillus, Mycoplasma or Staphylococcus which is capable of binding to the above tyrosine derivative in the presence of the above mutant TyrRS, and (C) a desired protein gene having a nonsense mutation at a desired site in animal cells to thereby incorporate the above tyrosine derivative into the nonsense mutation site in the above protein.

The invention discloses a regulation and control method of expression in a chimeric antigen receptor. An expression system enables coexpression of the chimeric antigen receptor, unnatural amino acid is coded to the corresponding UAG orthogonal unnatural amino acid arginyl tRNA and tRNA pair, the nonsense codon UAG is introduced into an amino acid sequence of the chimeric antigen receptor for coding the unnatural amino acid, the expression system is lack of the unnatural amino acid, the chimeric antigen receptor cannot be expressed, and when the expression system adds the unnatural amino acid, the chimeric antigen receptor can be completely expressed. The recombinant chimeric antigen receptor having unnatural amino acid-dependence keeps targeting and killing efficiency on CAR-T, and increases the controlling performance on two specific characters, a control switch is selected as a micromolecular compound, and the method provides an important approach for increasing the safety for cancer clinical treatment for CAR-T.

The invention discloses a preparation method of protein and polypeptide containing D-lysine. According to the invention, an expression gene of a target protein is introduced into a specific enzyme-digested multiple cloning site of an expression plasmid, and a target site of the expression gene is mutated into a TAG codon; a sequence of ph aminoacyl tRNA synthetase is introduced into another specific enzyme-digested multiple cloning site of the expression plasmid, such that a recombinant plasmid is obtained; a pAC-ph delta-AK3 plasmid is prepared; the pAC-ph delta-AK3 plasmid and the recombinant plasmid are cotransfected into a host, such that an expression system is obtained; D-lysine is added into a cultivation medium, and is used for inducing the expression system to express a target protein; the protein is purified, such that the target protein is obtained. According to the invention, a characteristic that lysyl tRNA synthetase and inhibitory type tRNA molecule pair originated frompyrococcushorikoshii can specifically select amber codon TAG is adopted, and D-lysine is introduced in a set position in double-carbonyl reductase. The introduction rate is close to 100%. The method has wide application values.

The present invention relates to a method for constructing a stable cell line carrying an orthogonal tRNA / aminoacyl tRNA synthetase, and the method stably integrates an orthogonal tRNA / aminoacyl tRNAsynthetase gene into cell genomes by double lentiviral stable transduction and plasmid stable transfection. The invention further relates to the stable cell line obtained by the method and the use ofthe stable cell line in the expression of a protein containing non-natural amino acids, and by utilizing unique reactive groups on the non-natural amino acids, the purpose of high-efficiency and specific protein modification can be achieved.

The invention includes a selective method of modifying the N-terminus of a protein using an aminoacyl tRNA transferase. In certain embodiments, the method comprises contacting a solution of the protein or peptide with a transferase and a derivative of a molecule, whereby the N-terminus of the protein or peptide is derivatized with the molecule.

The present invention relates to compositions and methods for treating conditions associated with angiogenesis. In particular the present invention relates to multi-unit complexes of tRNA synthetase fragments and uses thereof; diverse multi-unit complexes including a tRNA synthetase fragment; compositions and methods for modulating angiogenesis; polynucleotides encoding tRNA synthetase fragments and uses thereof; antibodies and epitopes specific to tRNA synthetase fragments; variants of tRNA synthetase fragments and uses thereof; methods for treating angiogenesis; methods for screening for anti-angiogenic agents; methods of modulating angiogenesis; kits for modulating angiogenesis; and business methods for modulating angiogenesis. Preferably the tRNA synthetase fragments are tryptophanyl tRNA synthetase fragments, and more preferably human tryptophanyl tRNA synthetase fragments.

This invention provides a method for producing proteins into which non-natural amino acids have been incorporated at desired positions involving the use of either prokaryote-derived or eukaryote-type aaRS* and prokaryotic cells or eukaryote-type as host cells, the method comprising steps of: (a) introducing genes encoding prokaryote-derived aminoacyl tRNA synthetase mutants having enhanced specificity to non-natural amino acids similar to given natural amino acids (compared with specificity to the natural amino acids) and tRNA genes for non-natural amino acids capable of binding to the non-natural amino acids in the presence of the prokaryote-derived aminoacyl tRNA synthetase mutants into prokaryotic cells that express genes encoding eukaryote-type aminoacyl tRNA synthetase having specificity to the given natural amino acids and tRNA genes capable of binding to the natural amino acids in the presence of eukaryote-type aminoacyl tRNA synthetase; and (b) knocking out genes encoding aminoacyl tRNA synthetase having specificity to the natural amino acids, which are inherent in the prokaryotic cells, and inherent tRNA genes capable of binding to the natural amino acids in the presence of the inherent aminoacyl tRNA synthetase.

Described are aminoacyl tRNA analogues which comprise a tRNA, and an amino acid which acts as an acceptor and donor substrate for ribosome-directed translation, thus, incorporating unusual monomers into non-standard polymers by the action of ribosomes. Also described are methods for producing such tRNA analogues; non-standard polymers; libraries of encoded polymers; methods of screening the libraries; and target members and their uses. A key advantage of synthesizing non-standard polymer libraries of the present invention with aminoacyl tRNA analogues is that large libraries of high complexity can be easily made and functional library members (e.g. novel drugs) readily identified.

The invention relates to a trypanosoma brucei phenylalanyl-tRNA synthetase preparation method, belonging to the technical field of biology and the preparation method comprises the following specific steps: separately designing primers, amplifying the alpha subunit and beta subunit genes of trypanosoma brucei phenylalanyl-tRNA synthetase; cloning the alpha subunit and beta subunit genes into prokaryotic expression vector pET21a(+), building pET21a(+)-PheRS alpha and pET21a(+)-PheRS beta expression vector; subcloning the operon of alpha subunit into pET21a(+)-PheRS beta, forming recombinant co-expression vector pET21a(+)-PheRS alpha beta; transforming the recombinant co-expression vector pET21a(+)-PheRS alpha beta into colibacillus BL21(DE3)RIPL, inducting expression with IPTG; collecting the supernate of the lysed bacteria solution, purifying proteins and obtaining the trypanosoma brucei phenylalanyl-tRNA synthetase. The invention builds the recombinant plasmid of the trypanosoma brucei phenylalanyl-tRNA synthetase (PheRS), and expresses and purifies trypanosoma brucei phenylalanyl-tRNA synthetase protein, thus laying a foundation for the screening of antiparasitic agent.

The invention includes a selective method of modifying the N-terminus of a protein using an aminoacyl tRNA transferase. In certain embodiments, the method comprises contacting a solution of the protein or peptide with a transferase and a derivative of a molecule, whereby the N-terminus of the protein or peptide is derivatized with the molecule.

The present invention relates to a novel function of lysyl tRNA synthetase (KRS) which enhances tumor cell migration and affects cancer metastasis via KRS's interaction with laminin receptor (67LR) by its translocation to membrane. More particularly, the present invention relates to a method for modulating cancer metastasis or migration, which comprises regulating intracellular levels of KRS; a composition for preventing or treating cancer; use of expression vector for inhibiting the expression of KRS; a method for preventing or treating cancer; use of an agent for inhibiting an activity of KRS; a method for screening an agent which modulates cancer metastasis or migration; and a method for screening an agent which inhibits the interaction of KRS with 67LR, by said novel function. Thus, KRS can modulate cancer metastasis or migration and furthermore, can modulate intracellular metabolism related to 67LR. The interaction between KRS and 67LR can be used effectively in treating, preventing and / or diagnosing of various diseases or disorders related to the interaction.

The invention relates to an aminoacyl-tRNA synthase mutant, which is an orthogonal aminoacyl-tRNA synthase. The amino acid sequence contained in the mutant is selected from the amino acid sequence shown in SEQIDNO:4 or its conservative variants or homologues with the same enzyme activity. The invention further provides a selenotyrosine translation system, which comprises (i) selenotyrosine, (ii) the orthogonal aminoacyl-tRNA synthetase, (iii) orthogonal tRNA and (iv) nucleic acid for encoding target protein, wherein the orthogonal aminoacyl-tRNA synthetase adopts the selenotyrosine for aminoacylation of the orthogonal tRNA preferentially, and the nucleic acid contains at least one selection codon for specifically recognizing the orthogonal tRNA. Moreover, the invention provides a method for modifying protease by utilizing the design of the selenotyrosine translation system and protease produced by encoding through the method.

The method comprising: a) obtaining a gene sequence codifying a naturally occurring aminoacyl-tRNA synthetase; b) engineering the gene codifying for said aminoacyl-tRNA synthetase, resulting into an aminoacyl-tRNA synthetase with a defective activity, with the proviso that the engineering does not affect the functionality of the catalytic site of the enzyme; c) cloning the gene resulting from step (b) in an expression vector; d) transforming isolated mammalian cells with the expression vector resulting from step (c); e) growing the recombinant cells resulting from step (d) in a nutrient medium under conditions which allow the expression of the engineered aminoacyl-tRNA synthetase, resulting the expression into cell death or a decrease in the rate of cell division; f) providing a substance to be tested to the medium resulting from step (e); and g) analyzing the resulting cell growth, wherein if there is an increase in cell growth, then the substance selectively inhibits the activity of the engineered aminoacyl-tRNA synthetase and does not affect to its cellular ortholog, resulting in that said substance is a candidate to drug.

It is intended to provide a method of chemically synthesizing an aminoacyl tRNA which is completely different from the existing methods, namely, a highly efficient and practically usable method for synthesizing an aminoacyl tRNA whereby any nonnatural amino acid can be conveniently aminoacylated without a need for genetic engineering techniques and detected without resort to a radioactive isotope. The above-described aminoacylation method comprises enclosing a tRNA and an amino acid in the vicinity of the micelle-water interface and bringing them close to each other to thereby react the same, or providing between them a peptide nucleic acid specifically binding to the tRNA as an antisense molecule and bringing them close to each other to thereby react the same.

The invention discloses a body fluid micromolecular metabolic marker of zebra fish infected bacteria, and the marker is at least one of glutamine, lactic acid, serine and glycine, preferably glutamine. The bacteria are selected from vibrio parahaemolyticus. The invention also discloses an application of glutamine in improving the immunity of zebra fish responding to vibrio parahaemolyticus infection, an application of glutamine in preparing medicines or health care products for improving the immunity of zebra fish responding to vibrio parahaemolyticus infection, an application of glutamine inregulation and control of galactose metabolism of zebra fish, and an application of glutamine in regulation and control of a biosynthetic metabolic pathway of aminoacyl tRNA of zebra fish. It is foundthrough experiments that glutamine plays an important role in zebra fish responding to bacterial infection, plays an extremely important role in zebra fish innate immune response, and can be used forresearch of metabolome functions, detection and treatment of zebra fish infected bacteria and the like. The marker has high reference value for research of metabonomics related to zebra fish responseto bacterial infection immune response.

The invention relates to a method for preparing trypanosoma brucei leucyl tRNA synthetase activated protein in the technical field of biology. The method comprises the following steps of: designing a specific primer and amplifying a trypanosoma brucei genome; recovering cut glue to obtain a trypanosoma brucei leucyl tRNA synthetase gene; connecting the gene obtained in the last step into a cloning vector to construct a gene cloning plasmid; subcloning the cloning plasmid into an expression vector, identifying, and converting into escherichia coli; cultivating the converted escherichia coli monoclone and inducing; and purifying expressed fusion protein to obtain the trypanosoma brucei leucyl tRNA synthetase activated protein. The leucyl tRNA synthetase activated protein can be used as a target for drug screening and is widely applied to the field of drug screening.