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1. 发明授权

US08785152B2 Process for production of non-natural protein having ester bond therein 失效
标题翻译：生产其中具有酯键的非天然蛋白质的方法
公开(公告)号：US08785152B2
公开(公告)日：2014-07-22
申请号：US12744322
申请日：2008-11-21
申请人： Shigeyuki Yokoyama , Kensaku Sakamoto , Tatsuo Yanagisawa , Takahito Mukai , Takatsugu Kobayashi
发明人： Shigeyuki Yokoyama , Kensaku Sakamoto , Tatsuo Yanagisawa , Takahito Mukai , Takatsugu Kobayashi
IPC分类号： C12P21/00 , C12N9/00 , C12N15/52 , C12N1/21
CPC分类号： C12N15/67 , C12N9/93 , C12P21/02
摘要： A non-natural protein having at least one ester bond in its polypeptide main chain is synthesized by using an in vivo translation system in a ribosome. The following components (a) to (c) are expressed in a cell or an cell extraction solution in the presence of an α-hydroxy acid: (a) an aminoacyl-tRNA synthetase which can activate the α-hydroxy acid; (b) suppressor tRNA which can bind to the α-hydroxy acid in the presence of the aminoacyl-tRNA synthetase; and (c) a gene encoding a desired protein having a nonsense mutation or a frame-shift mutation at a desired site.
摘要翻译：在其多肽主链中具有至少一个酯键的非天然蛋白质通过使用核糖体中的体内翻译系统来合成。以下组分（a）至（c）在α-羟基酸存在下在细胞或细胞提取溶液中表达：（a）可活化α-羟基酸的氨基酰-tRNA合成酶; （b）在氨基-tRNA合成酶存在下可结合α-羟基酸的抑制tRNA; 和（c）编码期望蛋白质的基因，所述蛋白质在期望的位点具有无义突变或帧位移突变。

2. 发明授权

US08735093B2 Mutant pyrrolysyl-tRNA synthetase, and method for production of protein having non-natural amino acid integrated therein by using the same 有权
标题翻译：突变型吡咯赖氨酰-tRNA合成酶，以及通过使用它们生成其中含有非天然氨基酸的蛋白质的方法
公开(公告)号：US08735093B2
公开(公告)日：2014-05-27
申请号：US12727037
申请日：2010-03-18
申请人： Shigeyuki Yokoyama , Kensaku Sakamoto , Tatsuo Yanagisawa , Takatsugu Kobayashi
发明人： Shigeyuki Yokoyama , Kensaku Sakamoto , Tatsuo Yanagisawa , Takatsugu Kobayashi
IPC分类号： C12P21/00 , C12N9/00 , C12N15/52 , C12N15/63
CPC分类号： C12N9/93 , C12P21/00 , C12P21/02
摘要： Method for incorporating a lysine derivative (particularly an Nε-benzyloxycarbonyl-lysine (Z-Lys) derivative) having useful functional group such as heavy atom, selenium, reactive functional group, fluorescent group or crosslinker, which is suitable as a non-natural amino acid, into a desired protein in a site-specific manner. A mutant pyrrolysyl-tRNA synthetase has substitution of at least one amino acid residue selected from tyrosine residue at position 306, leucine residue at position 309 and cysteine residue at position 348 each constituting a pyrrolysine-binding site in the amino acid sequence for pyrrolysyl-tRNA synthetase of SEQ ID NO:2. The substitution of the amino acid residue is: of tyrosine residue at position 306 by glycine or alanine residue, of leucine residue at position 309 by glycine or alanine residue, and/or of a cysteine residue at position 348 by valine, serine or alanine residue.
摘要翻译：引入适合作为非天然的有重要原子，硒，反应性官能团，荧光基团或交联剂等有用官能团的赖氨酸衍生物（特别是N，N-苄氧羰基赖氨酸（Z-Lys）衍生物）的方法氨基酸，以位点特异性方式转化成所需的蛋白质。突变型吡咯赖氨酰-tRNA合成酶具有至少一个氨基酸残基的取代，该氨基酸残基选自306位的酪氨酸残基，309位的亮氨酸残基和348位的半胱氨酸残基，每个构成吡咯赖氨酰-tRNA的氨基酸序列中的吡咯赖氨酸结合位点 SEQ ID NO：2的合成酶。氨基酸残基的取代是：位置306处的甘氨酸或丙氨酸残基的酪氨酸残基，309位的亮氨酸残基被甘氨酸或丙氨酸残基取代，和/或348位的半胱氨酸残基被缬氨酸，丝氨酸或丙氨酸残基取代。

3. 发明申请

US20130130313A1 METHOD OF SYNTHESIZING A SUPPRESSOR tRNA, DNA CONSTRUCT AND USE THEREOF FOR PRODUCING A NON-NATURAL AMINO ACID-INCORPORATED PROTEIN 有权
标题翻译：合成抑制剂tRNA的方法，DNA构建及其用于生产非天然氨基酸合成蛋白的方法
公开(公告)号：US20130130313A1
公开(公告)日：2013-05-23
申请号：US13460773
申请日：2012-04-30
申请人： Shigeyuki YOKOYAMA , Kensaku Sakamoto , Nobumasa Hino , Takahito Mukai , Takatsugu Kobayashi
发明人： Shigeyuki YOKOYAMA , Kensaku Sakamoto , Nobumasa Hino , Takahito Mukai , Takatsugu Kobayashi
IPC分类号： C12N15/85 , C12P21/00
CPC分类号： C12N15/85 , C12N15/67 , C12P19/34 , C12P21/00 , C12P21/02
摘要： There are provided a DNA construct comprising a suppressor tRNA gene of a non-eukaryote containing no internal promoter functioning in a eukaryotic cell, and a eukaryotic or bacteriophage promoter linked at the 5′ end of the tRNA gene, a method for synthesizing a suppressor tRNA by using the DNA construct, and a process for producing protein incorporating a non-natural amino acid by using the same.
摘要翻译：提供了包含在真核细胞中不具有内部启动子功能的非真核生物的抑制子tRNA基因的DNA构建体，以及在tRNA基因的5'末端连接的真核或噬菌体启动子，合成抑制子tRNA的方法通过使用DNA构建体，以及通过使用它们生产掺入非天然氨基酸的蛋白质的方法。

4. 发明申请

US20130095524A1 METHOD FOR CONSTRUCTING RECOMBINANT BACTERIUM FOR PRODUCING NON-NATIVE PROTEIN, AND UTILIZATION OF SAME 有权
标题翻译：用于构建用于生产非蛋白质的重组细菌的方法及其利用
公开(公告)号：US20130095524A1
公开(公告)日：2013-04-18
申请号：US13704391
申请日：2011-06-16
申请人： Shigeyuki Yokoyama , Takahito Mukai , Kensaku Sakamoto , Akiko Matsumoto
发明人： Shigeyuki Yokoyama , Takahito Mukai , Kensaku Sakamoto , Akiko Matsumoto
IPC分类号： C12N15/70
CPC分类号： C12N15/70 , C07K14/245 , C12N9/93 , C12N15/11 , C12N15/67 , C12P21/02 , Y02P20/52
摘要： The present invention provides a novel method of producing a recombinant bacterium for production of a non-natural protein, including: (1) expressing tRNA in a bacterium, which tRNA recognizes UAG codon; (2) expressing an aminoacyl-tRNA synthetase in the bacterium, which aminoacyl-tRNA synthetase acylates the tRNA with a non-natural amino acid or an α-hydroxy acid; (3) (i) introducing a DNA construct into the bacterium, which DNA construct is for expressing, in the absence of a release factor for terminating translation at UAG codon, a function of at least one gene selected from the group consisting of genes each of which loses its function when a gene that codes for the release factor is defective and/or introducing an alteration into said at least one gene in a chromosome of the bacterium, which alteration is for expressing the function of said at least one gene in the absence of the release factor; and (4) causing the gene that codes for the release factor in the bacterium to be defective.
摘要翻译：本发明提供了一种生产非天然蛋白质的重组细菌的新方法，包括：（1）在tRNA识别UAG密码子的细菌中表达tRNA; （2）在细菌中表达氨酰-tRNA合成酶，该氨基-tRNA合成酶用非天然氨基酸或α-羟基酸酰化tRNA; （3）（i）将DNA构建体引入细菌中，所述DNA构建体用于在不存在用于在UAG密码子下终止翻译的释放因子的情况下表达，所述DNA构建体用于表达至少一种选自下组的基因的功能：当编码释放因子的基因有缺陷和/或引入细菌染色体中的所述至少一个基因的改变时，其失去功能，所述改变用于在所述细菌的染色体中表达所述至少一种基因的功能不存在释放因子; 和（4）导致编码细菌释放因子的基因是有缺陷的。

5. 发明授权

US08293512B2 Polypeptide having activity of aminoacyl-tRNA synthetase and use thereof 有权
标题翻译：具有氨基-tRNA合成酶活性的多肽及其用途
公开(公告)号：US08293512B2
公开(公告)日：2012-10-23
申请号：US13180209
申请日：2011-07-11
申请人： Shigeyuki Yokoyama , Kensaku Sakamoto , Kenji Oki , Takahito Mukai
发明人： Shigeyuki Yokoyama , Kensaku Sakamoto , Kenji Oki , Takahito Mukai
IPC分类号： C12N9/00 , C07H21/04
CPC分类号： C12N9/93
摘要： A polypeptide according to the present invention includes: an altered polypeptide obtained by altering an ArgRS, a CysRS, a MetRS, a GlnRS, a GluRS, a LysRS, a TyrRS, or a TrpRS so that an unnatural amino acid is recognized; and an editing polypeptide derived from a PheRS, a LeuRS, an IleRS, a ValRS, an AlaRS, a ProRS, or a ThrRS, the editing polypeptide having been either inserted between a Rossman-fold N domain and a Rossman-fold C domain that exist in the altered polypeptide, or bound to an N terminal of the altered polypeptide. Thus provided are a new aaRS that exhibits high substrate specificity to an unnatural amino acid and a technique that involves the use of such an aaRS.
摘要翻译：根据本发明的多肽包括：通过改变ArgRS，CysRS，MetRS，GlnRS，GluRS，LysRS，TyrRS或TrpRS获得的改变的多肽，使得识别出非天然氨基酸; 和衍生自PheRS，LeuRS，IleRS，ValRS，AlaRS，ProRS或ThrRS的编辑多肽，所述编辑多肽已插入Rossman折叠N结构域和Rossman折叠C结构域之间，存在于改变的多肽中，或者与改变的多肽的N末端结合。因此提供了对非天然氨基酸表现出高底物特异性的新aaRS和涉及使用这种aaRS的技术。

6. 发明申请

US20110136168A1 PROCESS FOR PRODUCTION OF NON-NATURAL PROTEIN HAVING ESTER BOND THEREIN 失效
标题翻译：生产非蛋白质的非天然蛋白质的方法
公开(公告)号：US20110136168A1
公开(公告)日：2011-06-09
申请号：US12744322
申请日：2008-11-21
申请人： Shigeyuki Yokoyama , Kensaku Sakamoto , Tatsuo Yanagisawa , Takahito Mukai , Takatsugu Kobayashi
发明人： Shigeyuki Yokoyama , Kensaku Sakamoto , Tatsuo Yanagisawa , Takahito Mukai , Takatsugu Kobayashi
IPC分类号： C12P21/02
CPC分类号： C12N15/67 , C12N9/93 , C12P21/02
摘要： A non-natural protein having at least one ester bond in its polypeptide main chain is synthesized by using an in vivo translation system in a ribosome. The following components (a) to (c) are expressed in a cell or an cell extraction solution in the presence of an α-hydroxy acid: (a) an aminoacyl-tRNA synthetase which can activate the α-hydroxy acid; (b) suppressor tRNA which can bind to the α-hydroxy acid in the presence of the aminoacyl-tRNA synthetase; and (c) a gene encoding a desired protein having a nonsense mutation or a frame-shift mutation at a desired site.
摘要翻译：在其多肽主链中具有至少一个酯键的非天然蛋白质通过使用核糖体中的体内翻译系统来合成。以下组分（a）至（c）在α-羟基酸存在下在细胞或细胞提取溶液中表达：（a）可活化α-羟基酸的氨基酰-tRNA合成酶; （b）在氨基-tRNA合成酶存在下可结合α-羟基酸的抑制tRNA; 和（c）编码期望蛋白质的基因，所述蛋白质在期望的位点具有无义突变或帧位移突变。

7. 发明申请

US20090286311A1 DNA sequences for the expression of alloproteins 失效
标题翻译：用于表达alloproteins的DNA序列
公开(公告)号：US20090286311A1
公开(公告)日：2009-11-19
申请号：US12488136
申请日：2009-06-19
申请人： Shigeyuki Yokoyama , Mikako Shirouzu , Ayako Sakamoto , Kensaku Sakamoto
发明人： Shigeyuki Yokoyama , Mikako Shirouzu , Ayako Sakamoto , Kensaku Sakamoto
IPC分类号： C12N15/63 , C07H21/04
CPC分类号： C12N9/93 , A61K48/00
摘要： The present invention discloses an expression method for non-naturally-occurring amino acid-containing protein comprising: expressing in animal cells: (A) a mutant tyrosyl-tRNA synthetase that is a mutation of tyrosyl-tRNA synthetase originating in E. coli with an enhanced specificity for a non-naturally-occurring tyrosine derivative as compared with the specificity for tyrosine; (B) suppressor tRNA originating in Bacillus species, Mycoplasma species or Staphylococcus species of eubacteria and capable of binding with the tyrosine derivative in the presence of the mutant tyrosyl tRNA synthetase; and, (C) a desired protein gene that has undergone a nonsense mutation at a desired site; wherein, the tyrosine derivative is incorporated at the location of this nonsense mutation.
摘要翻译：本发明公开了一种非天然存在的含氨基酸蛋白质的表达方法，包括：在动物细胞中表达：（A）突变型酪氨酰-tRNA合成酶，其是源自大肠杆菌的酪氨酰-tRNA合成酶的突变，其具有与酪氨酸的特异性相比，非天然存在的酪氨酸衍生物的特异性增强; （B）源自芽孢杆菌属物种，支原体属或真菌属葡萄球菌属的抑制基因tRNA，能够在突变型酪氨酰tRNA合成酶存在下与酪氨酸衍生物结合; 和（C）在期望的位点已经经历了无义突变的期望的蛋白质基因; 其中，酪氨酸衍生物被掺入该无义突变的位置。

8. 发明申请

US20060234339A1 Method of expressing protein having unnatural amino acid integrated thereinto 失效
标题翻译：表达其中含有非天然氨基酸的蛋白质的方法
公开(公告)号：US20060234339A1
公开(公告)日：2006-10-19
申请号：US10532948
申请日：2003-10-31
申请人： Shigeyuki Yokoyama , Mikako Shirouzu , Ayako Sakamoto , Kensaku Sakamoto
发明人： Shigeyuki Yokoyama , Mikako Shirouzu , Ayako Sakamoto , Kensaku Sakamoto
IPC分类号： C12P21/06 , C07H21/04 , C12N9/22 , C12N5/06 , C12N15/09 , C07K14/47
CPC分类号： C12N9/93 , A61K48/00
摘要： A method of expressing a protein having an unnatural amino acid integrated thereinto characterized by comprising expressing (A) a mutant tyrosyl tRNA synthase which is a mutant of Escherichia coli-origin tyrosyl tRNA synthase and has an elevated specificity for a 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.
摘要翻译：一种表达具有整合在其中的非天然氨基酸的蛋白质的方法，其特征在于包括表达（A）突变型酪氨酰基tRNA合酶，其是大肠杆菌来源的酪氨酰基tRNA合酶的突变体，并且与非天然酪氨酸衍生物相比具有提高的特异性酪氨酸的特异性，（B）源自属于芽孢杆菌属，支原体属或葡萄球菌属的真细菌的抑制子tRNA，其在上述突变体TyrRS的存在下能够结合上述酪氨酸衍生物，和（C）期望的蛋白质基因在动物细胞中的所需位点处具有无义突变，从而将上述酪氨酸衍生物掺入上述蛋白质中的无义突变位点。

9. 发明授权

US06995249B1 Nucleic acid capable of binding specifically to Ras target protein 失效
标题翻译：能够特异性结合Ras靶蛋白的核酸
公开(公告)号：US06995249B1
公开(公告)日：2006-02-07
申请号：US09529397
申请日：1999-08-13
申请人： Shigeyuki Yokoyama , Ichiro Hirao , Kensaku Sakamoto
发明人： Shigeyuki Yokoyama , Ichiro Hirao , Kensaku Sakamoto
IPC分类号： C07H21/02 , C07H21/04
CPC分类号： C12N15/1135 , A61K38/00 , C12N2310/13
摘要： A novel nucleic acid (aptamer) which binds specifically to the target protein of Ras, more particularly, a novel RNA aptamer which binds specifically to Raf-1; a method for screening an RNA capable of binding specifically to the target protein of Ras which comprises selecting an RNA capable of binding to the target protein of Ras from a pool of RNAs having various base sequences; a method for regulating the signal transduction causing the proliferation or differentiation of cells by using the above-described nucleic acid; and medicinal compositions with the use of the same.
摘要翻译：特异性结合Ras靶蛋白的新型核酸（适配体），特别是与Raf-1特异性结合的新型RNA适体; 用于筛选能够特异性结合Ras的靶蛋白的RNA的方法，其包括从具有各种碱基序列的RNA库中选择能够结合Ras的靶蛋白的RNA; 通过使用上述核酸调节引起细胞增殖或分化的信号转导的方法; 和使用它们的药物组合物。

10. 发明申请

US20100304431A1 tRNA SYNTHESIS METHOD, NUCLEIC ACID, AMINOACYL tRNA SYNTHESIS METHOD, AND PROCESS FOR PRODUCTION OF PROTEIN INTRODUCED WITH UNNATURAL AMINO ACID 审中-公开
标题翻译： tRNA合成方法，核酸，氨基酸tRNA合成方法和生产引发氨基酸的蛋白质的方法
公开(公告)号：US20100304431A1
公开(公告)日：2010-12-02
申请号：US11997528
申请日：2006-08-02
申请人： Shigeyuki Yokoyama , Kensaku Sakamoto , Tatsuo Yanagisawa , Takatsugu Kobayashi
发明人： Shigeyuki Yokoyama , Kensaku Sakamoto , Tatsuo Yanagisawa , Takatsugu Kobayashi
IPC分类号： C12P21/02 , C12P19/34
CPC分类号： C12N9/93 , C12N15/67 , C12P19/34 , C12P21/02
摘要： The present invention relates to a process for producing a protein having an unnatural amino acid introduced therein, the process including: expressing in a eukaryotic cell an aminoacyl-tRNA synthetase, a nucleic acid having a sequence containing a eukaryote-derived tRNA nucleotide sequence linked to the 5′ end of a tRNA nucleotide sequence that is ligated with to an unnatural amino acid in the presence of the aminoacyl-tRNA synthetase, an unnatural amino acid, and a gene of a desired protein having a nonsense mutation at a predetermined position, to integrate the unnatural amino acid at the nonsense mutation position into the protein, thereby expressing a protein having an unnatural amino acid introduced therein.
摘要翻译：本发明涉及一种生产引入其中具有非天然氨基酸的蛋白质的方法，所述方法包括：在真核细胞中表达氨基-tRNA合成酶，具有包含与真核生物衍生的tRNA核苷酸序列连锁的序列的核酸在氨基-tRNA合成酶，非天然氨基酸和在预定位置具有无义突变的所需蛋白质的基因，在预定位置具有无义突变的基因，与非天然氨基酸连接的tRNA核苷酸序列的5'末端，将无义突变位置处的非天然氨基酸整合到蛋白质中，从而表达引入其中的非天然氨基酸的蛋白质。

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