50 results about "Aminoacylation" patented technology

Methods, compositions, kits, and apparatus are provided wherein the aminoacyl-tRNA synthetase system is used to analyze amino acids. The method allows very small devices for quantitative or semi-quantitative analysis of the amino acids in samples or in sequential or complete proteolytic digestions. The methods can be readily applied to the detection and/or quantitation of one or more primary amino acids by using cognate aminoacyl-tRNA synthetase and cognate tRNA. The basis of the method is that each of the 20 synthetases and/or a tRNA specific for a different amino acid is separated spatially or differentially labeled. The reactions catalyzed by all 20 synthetases may be monitored simultaneously, or nearly simultaneously, or in parallel. Each separately positioned synthetase or tRNA will signal its cognate amino acid. The synthetase reactions can be monitored using continuous spectroscopic assays. Alternatively, since elongation factor Tu:GTP (EF-Tu:GTP) specifically binds all AA-tRNAs, the aminoacylation reactions catalyzed by the synthetases can be monitored using ligand assays. Microarrays and microsensors for amino acid analysis are provided. Additionally, amino acid analysis devices are integrated with protease digestions to produce miniaturized enzymatic sequenators capable of generating either N- or C-terminal sequence and composition data for a protein or peptide. The possibility of parallel processing of many samples in an automated manner is discussed.

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.

The invention relates to an aminoacyl-tRNA synthetase mutant which is orthogonal aminoacyl-tRNA synthetase. The amino acid sequence of the synthetase is selected from a group composed of an amino acid sequence presented in SEQ ID NO:4 and conservative variants of the amino acid sequence presented in SEQ ID NO:4, and the conservative variants have enzyme activity the same as the amino acid sequence presented in SEQ ID NO:4. The invention further provides a 3,5-difluoro-tyrosine translation system which comprises (i) 3,5-difluoro-tyrosine, (ii) the orthogonal aminoacyl-tRAN synthetase, (iii) orthogonal tRNA and (iv) nucleic acid of encoded target protein, wherein the orthogonal aminoacyl-tRAN synthetase carries out prior aminoacylation on the orthogonal tRNA through the 3,5-difluoro-tyrosine, and the nucleic acid contains at least one selective codon for specific recognition of the orthogonal tRNA.

The invention relates to an aminoacyl-tRNA synthetase mutant which has an amino acid sequence selected from a group comprising an amino acid sequence as shown in SEQ ID NO:4 and conservative variants of the amino acid sequence as shown in SEQ ID NO:4, wherein the conservative variants have the same enzymatic activity as the amino acid sequence as shown in SEQ ID NO:4. The The invention also provides a translation system which comprises: (i) 8-hydroxyquinoline alanine or its structure analogs; (ii) an orthogonal aminoacyl-tRNA synthetase of the invention; (iii) orthogonal tRNA, wherein the orthogonal aminoacyl-tRNA synthetase realizes prior aminoacylation of the orthogonal tRNA by the 8-hydroxyquinoline alanine or its structure analogs; and (iv) nucleic acid coding target protein, wherein the nucleic acid contains at least one selection codon specifically recognized by the orthogonal tRNA. Finally, the invention provides a tyrosine phenol lyase mutant which can catalyze 8-hydroxyquinoline to generate 8-hydroxyquinoline alanine, and has an amino acid sequence as shown in SEQ ID NO:5.

The present invention provides catalytic RNA molecules having cis or trans aminoacylation activity. The catalytic RNA molecules having cis aminoacylation activity comprise a catalytic domain and an aminoacylation domain. The catalytic RNA molecules having trans aminoacylation activity only have the catalytic domain. A method is provided for constructing and screening of these molecules. These molecules are suitable for aminoacylating with specific amino acids.

The invention provides an acrylyl lysine translation system which dopes acrylyl lysine into target protein by using an orthogonal tRNA, an orthogonal acrylyl lysine aminoacyl tRNA synthetase and their combination, and also provides a method for specifically doping acrylyl lysine into target protein at a fixed point by using the translation system. The acrylyl lysine translation system comprises: (i) acrylyl lysine; (ii) the orthogonal acrylyl lysine aminoacyl tRNA synthetase; (iii) the orthogonal tRNA, wherein the orthogonal acrylyl lysine aminoacyl tRNA synthetase is used for preferential aminoacylation of the orthogonal tRNA by the acrylyl lysine; and (iv) nucleic acid coding the target protein, wherein the nucleic acid contains at least one selection codon specifically recognized by the orthogonal tRNA. The invention also relates to an application of the mutant protein doped with the acrylyl lysine.

The invention relates to an aminoacyl-tRNA synthetase mutant. The aminoacyl-tRNA synthetase mutant has an amino acid sequence composed of amino acids shown in the formula of SEQ ID NO.2 and their conservative mutants. The invention provides a 3-imidazolyl tyrosine translation system for specific and fixed-point insertion of 3-imidazolyl tyrosine (imiTyr) into a target protein by utilization of an orthogonal tRNA, an orthogonal aminoacyl-tRNA synthetase and their complex, and also provides a method for specific and fixed-point insertion of the 3-imidazolyl tyrosine into the target protein by the 3-imidazolyl tyrosine translation system. The 3-imidazolyl tyrosine translation system comprises 1), 3-imidazolyl tyrosine, 2), the orthogonal aminoacyl-tRNA synthetase, 3), the orthogonal tRNA, wherein the orthogonal aminoacyl-tRNA synthetase is obtained by preferential aminoacylation of the orthogonal tRNA by the 3-imidazolyl tyrosine, and 4), a nucleic acid for encoding a target protein, wherein the nucleic acid contains at least one selection codons specifically recognized by the orthogonal tRNA. The invention also relates to a method for specific and fixed-point insertion of 3-imidazolyl tyrosine in Myoglobin (Mb) so that the Myoglobin has oxidase activity, and a use of the method.

The present invention provides modified aminoacyl-tRNA synthetases (ARSs) having increased reactivity with N-methyl amino acids compared to natural aminoacyl-tRNA synthetases. The modified aminoacyl-tRNA synthetases according to the present invention can aminoacylate tRNAs with their corresponding N-methyl-substituted amino acids such as N-methyl-phenylalanine, N-methyl-valine, N-methyl-serine, N-methyl-threonine, N-methyl-tryptophan, and N-methyl-leucine more efficiently than natural aminoacyl-tRNA synthetases. The present invention enables a more efficient production of polypeptides containing N-methyl amino acids.

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.

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.

Provided is a method for synthesizing a protein, into which a nucleobase amino acid (NBA) is introduced at a desired position, that comprises: a step for preparing mRNA into which a modified codon is inserted at a desired position downstream of an initiation codon; and a step for translating the aforesaid mRNA into a protein in the presence of tRNA, said tRNA recognizing the modified codon and being acylated with the NBA. Also provided is a ribozyme that catalyzes the aminoacylation of tRNA and comprises two RNA molecules.

In the prior, the conventional 3-(1-aminoethyl)-2-cyclobutyl-2-hydro-isoquinolin-1-one compound synthesis method has disadvantages of low yield, harsh conditions, and difficult purification. Based on the disadvantages in the prior art, the present invention provides a synthesis route for synthesizing a 3-(1-aminoethyl)-2-cyclobutyl-2-hydro-isoquinolin-1-one compound, wherein o-toluoyl chloride is adopted as a starting raw material, and is subjected to different aminoacylation, butyl lithium is adopted to carry out dehydrogenation on hydrogen on the methyl group, the resulting material is substituted with a protected amino acid ester, and finally a phosphorusoxychloride one-pot cooking method is adopted to obtain the final product, such that the whole process is easy to operate, the product is easy to purify, the method is suitable for industry, and raw material cost is substantially reduced.