公开(公告)号：US20190194286A1

公开(公告)日：2019-06-27

申请号：US16327193

申请日：2016-07-29

申请人： Shigeyuki YOKOYAMA

发明人： Shigeyuki YOKOYAMA ,  Takehiro SHINODA ,  Kaori ITO ,  Naoko NOMURA ,  Yoshiko KATSURA ,  Tomomi SOMEYA ,  Mikako SHIROUZU ,  Tetsuya HORI ,  Yoshihiro NAKAMURA ,  Hiroaki TANABE

IPC分类号： C07K14/705 ,  C12N9/64

CPC分类号： C07K14/705 ,  C12N9/6478 ,  C12N15/09 ,  C12P21/02 ,  C12Y304/23046

摘要： In order to provide a membrane protein production method which does not require the step of solubilizing a membrane protein and which allows the membrane protein having an excellent quality to be obtained with a high yield, a method in accordance with an embodiment of the present invention includes: a step (a) of preparing a reaction solution for cell-free protein synthesis, the reaction solution containing (i) a template nucleic acid which encodes the membrane protein, (ii) a lipid, and (iii) a detergent which is contained at a concentration equal to or higher than a critical micelle concentration; and a step (b) of synthesizing the membrane protein while the concentration of the detergent in the reaction solution is maintained at a concentration equal to or higher than a critical micelle concentration.

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。

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 。

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。

公开(公告)号：US20130122506A1

公开(公告)日：2013-05-16

申请号：US13642111

申请日：2011-04-21

申请人： Ichiro Hirao ,  Michiko Hirao ,  Shigeyuki Yokoyama ,  Tsuneo Mitsui ,  Rie Yamashige

发明人： Ichiro Hirao ,  Michiko Hirao ,  Shigeyuki Yokoyama ,  Tsuneo Mitsui ,  Rie Yamashige

IPC分类号： G01N21/64

CPC分类号： G01N21/6486 ,  C07H19/044 ,  C12Q1/6818 ,  C12Q2525/117

摘要： It is an object of the present invention to provide quenching or fluorescent nucleic acid base analogs and applications thereof. The quencher of the present invention has a 2-nitropyrrole structure represented by Formula I:[Formula 1] (in Formula I, R1 and R2 are groups independently selected from the group consisting of: ribose and deoxyribose; hydrogen, hydroxyl and SH groups, and halogens; substituted or unsubstituted alkyl, alkenyl, and alkynyl groups each having 2 to 10 carbon atoms; one or more five-membered heterocyclic rings, one or more six-membered heterocyclic rings, and one or more fused heterocyclic rings, these heterocylic rings containing nitrogen or sulfur, and one or more aromatic rings; sugars, sugar chains, amino acids, and peptides; and fluorescent molecules linked via linkers).

摘要翻译： 本发明的目的是提供淬灭或荧光核酸碱基类似物及其应用。 本发明的猝灭剂具有由式I表示的2-硝基吡咯结构：[式1]（式I中，R 1和R 2分别独立地选自：核糖和脱氧核糖;氢，羟基和SH基团， 卤素;取代或未取代的各自具有2-10个碳原子的烷基，烯基和炔基;一个或多个五元杂环，一个或多个六元杂环和一个或多个稠合杂环，这些杂环 含有氮或硫，以及一个或多个芳环;糖，糖链，氨基酸和肽;以及通过接头连接的荧光分子）。

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。

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。

公开(公告)号：US20120245340A1

公开(公告)日：2012-09-27

申请号：US13500303

申请日：2010-10-06

申请人： Ichiro Hirao ,  Michiko Hirao ,  Shigeyuki Yokoyama ,  Tsuneo Mitsui

发明人： Ichiro Hirao ,  Michiko Hirao ,  Shigeyuki Yokoyama ,  Tsuneo Mitsui

IPC分类号： C07H19/20 ,  C07H21/02 ,  C07H19/173 ,  C07H21/04 ,  C12P19/34 ,  C07H19/048

CPC分类号： C07D471/04 ,  C07D473/32 ,  C07H19/173 ,  C07H19/23 ,  C09K11/06 ,  C09K2211/1074

摘要： The present invention relates to novel unnatural fluorescent nucleic acid bases, that is, a purine base, a 1-deazapurine base, and a 1,7-deazapurine base each having a functional group which consists of two or more heterocyclic moieties linked together, at the 6-position thereof (the 6-position of purine ring). The present invention also relates to a compound containing the unnatural base, a derivative thereof, and a nucleic acid containing a nucleotide having the unnatural base. The present invention also relates to a method of preparing the nucleic acid. The unnatural base of the present invention has excellent fluorescence characteristics and also has excellent properties as a universal base.

摘要翻译： 本发明涉及新型非天然荧光核酸碱基，即嘌呤碱，1-脱氮嘌呤碱和1,7-deazapurine碱，其各自具有由两个或多个连接在一起的杂环基部分组成的官能团，在 其6-位（嘌呤环的6-位）。 本发明还涉及含有非天然碱，其衍生物和含有具有非天然碱基的核苷酸的核酸的化合物。 本发明还涉及制备核酸的方法。 本发明的非天然碱具有优异的荧光特性，也具有优异的作为通用基材的特性。

公开(公告)号：US08178301B2

公开(公告)日：2012-05-15

申请号：US12318344

申请日：2008-12-24

申请人： Shigeyuki Yokoyama ,  Ryuya Fukunaga ,  Shun-ichi Sekine

发明人： Shigeyuki Yokoyama ,  Ryuya Fukunaga ,  Shun-ichi Sekine

IPC分类号： C12Q1/68 ,  C12N9/00 ,  C12N15/63 ,  C12N1/21

CPC分类号： C12P21/02 ,  C12N9/93

摘要： A mutant SepRS which is suitable for a site-specific introduction of phosphoserine into a protein is prepared by analyzing the structure and functions of a phosphoseryl-tRNA synthetase (SepRS) derived from an archaebacterium. A mutant SepRS composed of an amino acid sequence depicted in SEQ ID NO:2, in which any one or more of glutamic acids at position-418 and position-420 and threonine at position-423 are substituted with other amino acid, and having enhanced binding affinity with a suppressor tRNA as compared with a wild type phosphoseryl-tRNA synthetase (SepRS) composed of an amino acid sequence depicted in SEQ ID NO:2 is provided.

摘要翻译： 通过分析源自古细菌的磷酰基-tRNA合成酶（SepRS）的结构和功能，制备适合于将磷酸丝氨酸位点特异性引入蛋白质的突变体SepRS。 由SEQ ID NO：2所示的氨基酸序列构成的突变体SepRS，其中418位和420位的谷氨酸和423位的苏氨酸中的任何一种或多种被其它氨基酸取代，并具有增强的 提供了与由SEQ ID NO：2所示的氨基酸序列组成的野生型磷酰基-tRNA合成酶（SepRS）相比，与抑制子tRNA的结合亲和性。

公开(公告)号：US20110300575A1

公开(公告)日：2011-12-08

申请号：US13211280

申请日：2011-08-16

申请人： Hiroaki IMATAKA ,  Satoshi Mikami ,  Shigeyuki Yokoyama

发明人： Hiroaki IMATAKA ,  Satoshi Mikami ,  Shigeyuki Yokoyama

IPC分类号： C12P21/00 ,  C12N9/14

CPC分类号： C12P21/02 ,  C07K14/4702

摘要： Prepared is an extract composition having an improved protein synthetic activity in a cell-free protein synthesis system using a mammalian cultured cell extract. An eukaryotic translation initiation factor and/or translational regulator are added to a cell-free protein synthesis system comprising an extract prepared from cultured mammalian cells and a template mRNA. These factors are one or more selected from the group consisting of eukaryotic translation initiation factors 4E (eIF4E), 2 (eIF2) and 2B (eIF2B), and eukaryotic translational regulator p97.

摘要翻译： 制备的是在使用哺乳动物培养细胞提取物的无细胞蛋白质合成系统中具有改进的蛋白质合成活性的提取物组合物。 将真核翻译起始因子和/或翻译调节子添加到包含由培养的哺乳动物细胞和模板mRNA制备的提取物的无细胞蛋白质合成系统中。 这些因子是选自真核翻译起始因子4E（eIF4E），2（eIF2）和2B（eIF2B）以及真核翻译调节子p97中的一种或多种。