JP2019047763A - Rhodopsin phosphodiesterase - Google Patents
Rhodopsin phosphodiesterase Download PDFInfo
- Publication number
- JP2019047763A JP2019047763A JP2017174601A JP2017174601A JP2019047763A JP 2019047763 A JP2019047763 A JP 2019047763A JP 2017174601 A JP2017174601 A JP 2017174601A JP 2017174601 A JP2017174601 A JP 2017174601A JP 2019047763 A JP2019047763 A JP 2019047763A
- Authority
- JP
- Japan
- Prior art keywords
- pde
- light
- cells
- camp
- phosphodiesterase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 title claims abstract description 12
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 title claims abstract description 12
- NCYCYZXNIZJOKI-IOUUIBBYSA-N 11-cis-retinal Chemical compound O=C/C=C(\C)/C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-IOUUIBBYSA-N 0.000 title claims abstract description 9
- 102000004330 Rhodopsin Human genes 0.000 title claims abstract description 9
- 108090000820 Rhodopsin Proteins 0.000 title claims abstract description 9
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 claims abstract description 24
- 230000000694 effects Effects 0.000 claims description 10
- 230000015556 catabolic process Effects 0.000 claims description 4
- 238000006731 degradation reaction Methods 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 29
- 125000004122 cyclic group Chemical group 0.000 abstract description 18
- 210000005260 human cell Anatomy 0.000 abstract description 6
- 210000004962 mammalian cell Anatomy 0.000 abstract description 5
- 230000003287 optical effect Effects 0.000 abstract description 5
- 241000662257 Salpingoeca rosetta Species 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 241001134734 Choanoflagellida Species 0.000 abstract description 2
- 230000001413 cellular effect Effects 0.000 abstract description 2
- 230000002829 reductive effect Effects 0.000 abstract description 2
- 230000011664 signaling Effects 0.000 abstract description 2
- 230000002068 genetic effect Effects 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 35
- 102000004169 proteins and genes Human genes 0.000 description 23
- ZOOGRGPOEVQQDX-UHFFFAOYSA-N cyclic GMP Natural products O1C2COP(O)(=O)OC2C(O)C1N1C=NC2=C1NC(N)=NC2=O ZOOGRGPOEVQQDX-UHFFFAOYSA-N 0.000 description 21
- 238000000034 method Methods 0.000 description 15
- 201000010099 disease Diseases 0.000 description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 10
- 230000007246 mechanism Effects 0.000 description 9
- 239000013598 vector Substances 0.000 description 8
- 230000003247 decreasing effect Effects 0.000 description 7
- 230000003834 intracellular effect Effects 0.000 description 7
- 238000004020 luminiscence type Methods 0.000 description 7
- 241000282414 Homo sapiens Species 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 241000282412 Homo Species 0.000 description 5
- 210000004748 cultured cell Anatomy 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 238000000354 decomposition reaction Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000031146 intracellular signal transduction Effects 0.000 description 4
- 230000001678 irradiating effect Effects 0.000 description 4
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 208000006673 asthma Diseases 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 208000030603 inherited susceptibility to asthma Diseases 0.000 description 3
- 238000001638 lipofection Methods 0.000 description 3
- 210000002460 smooth muscle Anatomy 0.000 description 3
- NCYCYZXNIZJOKI-UHFFFAOYSA-N vitamin A aldehyde Natural products O=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- NCYCYZXNIZJOKI-OVSJKPMPSA-N Retinaldehyde Chemical compound O=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-OVSJKPMPSA-N 0.000 description 2
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 2
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 2
- 102000030621 adenylate cyclase Human genes 0.000 description 2
- 108060000200 adenylate cyclase Proteins 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229940095074 cyclic amp Drugs 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 108091008695 photoreceptors Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- RQFCJASXJCIDSX-UHFFFAOYSA-N 14C-Guanosin-5'-monophosphat Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(O)=O)C(O)C1O RQFCJASXJCIDSX-UHFFFAOYSA-N 0.000 description 1
- 125000001572 5'-adenylyl group Chemical group C=12N=C([H])N=C(N([H])[H])C=1N=C([H])N2[C@@]1([H])[C@@](O[H])([H])[C@@](O[H])([H])[C@](C(OP(=O)(O[H])[*])([H])[H])([H])O1 0.000 description 1
- AEOBEOJCBAYXBA-UHFFFAOYSA-N A2P5P Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1OP(O)(O)=O AEOBEOJCBAYXBA-UHFFFAOYSA-N 0.000 description 1
- 239000013607 AAV vector Substances 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000190909 Beggiatoa Species 0.000 description 1
- 241000235416 Blastocladiella emersonii Species 0.000 description 1
- 108010035848 Channelrhodopsins Proteins 0.000 description 1
- 101710095468 Cyclase Proteins 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000097097 Laurella Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000499883 Solaria <angiosperm> Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 210000003792 cranial nerve Anatomy 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 150000002266 vitamin A derivatives Chemical class 0.000 description 1
Abstract
Description
本発明は、光スイッチ型ホスホジエステラーゼに関する。 The present invention relates to a photoswitched phosphodiesterase.
生命現象を光で操作する技術は光遺伝学（オプトジェネティクス）と呼ばれ、近年飛躍的な発展を遂げている。例えばチャネルロドプシンと呼ばれる光スイッチタンパク質を神経細胞へ導入することで、その神経細胞の興奮を光照射で瞬時に誘起することが可能になった。このような光スイッチ型分子を用いた神経活動の光操作技術は、記憶形成メカニズムといった脳神経機能の解明に大きく貢献している。 The technology to manipulate life phenomena with light is called optogenetics, and has achieved rapid development in recent years. For example, by introducing a light switch protein called channelrhodopsin into neurons, it has become possible to instantaneously induce excitation of the neurons by light irradiation. Optical manipulation technology of neural activity using such optical switch type molecule greatly contributes to elucidation of cranial nerve function such as memory formation mechanism.
一方、細胞内シグナル伝達物質濃度を光操作するための酵素活性を示す光スイッチ分子は限られている。その分子として非特許文献１に、ミドリムシ由来の光スイッチ型アデニル酸シクラーゼ（ＰＡＣ）が開示されている。また、非特許文献２に菌類由来の光スイッチ型グアニルサンシクラーゼ（ＢｅＧＣ１）が知られている。 On the other hand, photoswitch molecules that exhibit enzyme activity for light manipulation of intracellular signal transduction substance concentration are limited. Non-Patent Document 1 discloses a light-switched adenylate cyclase (PAC) derived from green leaf as its molecule. Further, Non-Patent Document 2 discloses a photoswitch type guanyl sancyclase (BeGC1) derived from fungi.
これらタンパク質分子は、細胞内シグナル伝達物質であるサイクリックＡＭＰ、サイクリックＧＭＰ等の環状ヌクレオチド（以下、ｃＡＭＰ、ｃＧＭＰと言う場合がある）を合成することができる光スイッチ型酵素分子である。これら分子をヒトなどの細胞や組織へ導入することにより、光照射によって細胞内のｃＡＭＰ、ｃＧＭＰ量を高めることができる。 These protein molecules are photoswitched enzyme molecules capable of synthesizing cyclic nucleotides (hereinafter, sometimes referred to as cAMP and cGMP) such as intracellular signal transduction substances cyclic AMP and cyclic GMP. By introducing these molecules into cells or tissues such as human, the amount of cAMP and cGMP in cells can be increased by light irradiation.
ところが、細胞内のｃＡＭＰ、ｃＧＭＰ量を下げるための光スイッチ分子は自然界から発見されておらず、現状では薬剤添加による方法が一般的である。しかしながら薬剤を使用した場合、その薬剤は標的細胞以外にも効果を及ぼしてしまうという点が第１の問題である。第２の問題として薬剤投与後、その効果が奏するまでに時間を要すること、また１度薬剤を投与すると、それを除去して投与前の状態に戻すのにも長時間を要する。このように薬剤を用いた研究では限界がある。従って、標的細胞特異的、かつ短時間での細胞内のｃＡＭＰ、ｃＧＭＰ量の抑制効果の検証が困難である。
However, optical switch molecules for reducing the amount of cAMP and cGMP in cells have not been found in nature, and at present, methods by drug addition are generally used. However, when using a drug, the first problem is that the drug exerts an effect on other than the target cells. The second problem is that after drug administration, it takes time for its effect to be effective, and once it is administered, it takes a long time to remove it and return it to the state before administration. Thus, there are limitations in drug research. Therefore, it is difficult to verify the inhibitory effect of cAMP and cGMP in the target cell specific and in a short time.
本発明の課題は上記のような従来の問題を解決し、ヒトの細胞や組織内の環状ヌクレオチド濃度を光によって迅速に抑制する光スイッチ分子を提供することである。 An object of the present invention is to solve the conventional problems as described above, and to provide a light switch molecule that rapidly suppresses the cyclic nucleotide concentration in human cells and tissues with light.
光照射によってｃＡＭＰ、ｃＧＭＰを分解するロドプシンホスホジエステラーゼ（以下、Ｒｈ−ＰＤＥと言う場合がある）をヒトなどの哺乳類細胞、組織へ導入する。 Rhodopsin phosphodiesterase (hereinafter sometimes referred to as Rh-PDE) that degrades cAMP and cGMP by light irradiation is introduced into mammalian cells such as humans and tissues.
薬剤ではなく、光照射されたＲｈ−ＰＤＥによって、人などの哺乳類細胞内の環状ヌクレオチド濃度を迅速に抑制するという効果を奏する。細胞内の環状ヌクレオチド濃度の低下は、様々な疾患の原因となることが知られている。従って例えば、Ｒｈ−ＰＤＥを健常な動物の細胞や組織に導入すれば、光照射で環状ヌクレオチド濃度を低下させることでその動物を疾患状態に一過的に陥らせることが可能となり、革新的な疾患研究手法となる可能性がある。 Irradiated Rh-PDE, not a drug, has the effect of rapidly suppressing the cyclic nucleotide concentration in mammalian cells such as humans. Decreased intracellular cyclic nucleotide concentrations are known to cause various diseases. Thus, for example, if Rh-PDE is introduced into cells or tissues of a healthy animal, it is possible to transiently bring the animal into a disease state by reducing the cyclic nucleotide concentration by light irradiation, which is innovative It may be a disease research method.
本発明者らは、襟べん毛虫（襟べん毛虫門、カラエリヒゲムシ属 学名Salpingoeca rosetta）という微生物において、光受容体であるロドプシンと、ホスホジエステラーゼが連続した遺伝子コードをゲノム中に持つことに着目して本発明を完成した。 The present inventors focused attention on the fact that, in a microbe named Laurella flagellum (Solaria phylum, Salpingoeca rosetta), the genome contains the gene code of consecutive rhodopsin, which is a photoreceptor, and phosphodiesterase. The present invention has been completed.
すなわち、その遺伝子がコードするたんぱく質（Ｒｈ−ＰＤＥ）が、光に反応して環状ヌクレオチド分解活性を示すこと見出した。そして、このたんぱく質を人の培養細胞に導入したところ、光照射によって細胞内の環状ヌクレオチド濃度を抑制することができた。 That is, it has been found that the protein encoded by that gene (Rh-PDE) exhibits cyclic nucleotide degradation activity in response to light. Then, when this protein was introduced into human cultured cells, it was possible to suppress the cyclic nucleotide concentration in the cells by light irradiation.
ここで、襟べん毛虫（Ｃｈｏａｎｏｍｏｎａｄａ、コアノモナダ）とは、体長数ミクロンの単細胞生物で１本のべん毛を持つ。単細胞生物ではあるが、進化的には多細胞生物に最も近いとされる。ロドプシンとは、発色団としてレチナールを結合しており、光が当たるとイオン輸送などの機能を発揮する光受容型膜たんぱく質である。また、ホスホジエステラーゼ（ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ）とは、環状ヌクレオチド加水分解酵素のことであり、人では１１種類のファミリーが知られており、その基質特異性や、活性調節の仕組みが異なっている。ヒトなどの哺乳類がもつホスホジエステラーゼは光で直接働くことはできない。 As used herein, the term “Choanomonada” refers to a single-celled organism having a length of several microns and having one flagella. Although it is a unicellular organism, it is evolutionarily closest to multicellular organisms. Rhodopsin is a photoreceptor membrane protein that binds retinal as a chromophore and exhibits functions such as ion transport when exposed to light. Moreover, phosphodiesterase (phosphodiesterase) is a cyclic nucleotide hydrolase, and in humans, 11 types of families are known, and their substrate specificities and mechanisms of activity regulation differ. Phosphodiesterases possessed by mammals such as humans can not work directly with light.
ここで、環状ヌクレオチドとは、サイクリックＡＭＰ、サイクリックＧＭＰ（以下、ｃＡＭＰ、ｃＧＭＰと言う場合がる）などの環状エステル結合を持つヌクレオチドのことであり、細胞内シグナル情報伝達においてセカンドメッセンジャー（二次情報伝達物質）として働いている。なお、ｃＡＭＰ、ｃＧＭＰは環状アデノシン一リン酸、および環状グアノシン一リン酸の略称である。 Here, a cyclic nucleotide is a nucleotide having a cyclic ester bond such as cyclic AMP or cyclic GMP (hereinafter, may be referred to as cAMP or cGMP), and is a second messenger in intracellular signal transduction. It works as the next information carrier). CAMP and cGMP are abbreviations of cyclic adenosine monophosphate and cyclic guanosine monophosphate.
ｃＡＭＰやｃＧＭＰは、細胞内シグナル情報伝達物質であり、細胞内で働くさまざまな酵素の機能を調節している。環状ヌクレオチドは必要に応じて合成され、ＰＤＥにより分解されることで、細胞内での濃度が適切に保たれている。 cAMP and cGMP are intracellular signal transducers, and regulate the functions of various enzymes that work in cells. Cyclic nucleotides are optionally synthesized and degraded by PDE to maintain the intracellular concentration appropriately.
環状ヌクレオチドは呼吸器系疾患や認知症、網膜色素変性症などの疾患にも深く関与するため、Ｒｈ−ＰＤＥを応用した細胞内シグナル伝達の光操作法を確立することによって、これら疾患メカニズムの解明にも貢献することができる。 Since cyclic nucleotides are deeply involved in diseases such as respiratory diseases, dementia, and retinitis pigmentosa, elucidation of the mechanism of these diseases by establishing a light manipulation method of intracellular signal transduction applying Rh-PDE. Can also contribute.
例えば、気管支喘息は気道が狭くなることで粘膜泌物がたまり、軌道に炎症がおこることで発症する。気道が狭くなるのは気道を構成する気管支平滑筋内のｃＡＭＰ量が減少することで平滑筋が収縮するためである。ゆえに例えばＲｈ−ＰＤＥを健常マウスの平滑筋に導入した場合、光照射でｃＡＭＰが分解され、人為的かつ迅速に気管支喘息の状態を発現することが可能となる。このように疾患状態を光照射で自在に作り出すことができれば、疾患メカニズムの解明、さらには治療法の確立に貢献すると期待される。 For example, bronchial asthma develops when mucosal airways accumulate due to narrowing of the airway and inflammation occurs on the trajectory. The airway narrows because the smooth muscle contracts by decreasing the amount of cAMP in bronchial smooth muscle that constitutes the airway. Therefore, for example, when Rh-PDE is introduced into the smooth muscle of a healthy mouse, light irradiation degrades cAMP, and it becomes possible to artificially and rapidly develop a bronchial asthma condition. Thus, if the disease state can be freely created by light irradiation, it is expected to contribute to the elucidation of the disease mechanism and the establishment of a treatment.
以下、図面を参照しつつ本発明の実施の形態について説明する。本発明は、以下の実施形態に限定されるものではなく、発明の範囲を逸脱しない限りにおいて、変更、修正、改良を加え得るものである。なお、説明が重複する箇所については、適宜説明を省略する場合があるが、発明の要旨を限定するものではない。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is not limited to the following embodiments, and changes, modifications, and improvements can be made without departing from the scope of the invention. In addition, although description may be suitably abbreviate | omitted about the location where description overlaps, it does not limit the summary of invention.
本発明で使用されるＲｈ−ＰＤＥタンパク質の調製は、例えば、Ｒｈ−ＰＤＥタンパク質をコードする遺伝子配列を含む発現ベクターが導入された形質転換体を使用することで行うことができる。例えば、まず、この形質転換体を適宜の条件で培養し、遺伝子配列がコードするＲｈ−ＰＤＥタンパク質を合成させる。そして、合成されたタンパク質を形質転換体又は培養液から回収することにより、本発明のＲｈ−ＰＤＥタンパク質を得ることができる。 The Rh-PDE protein used in the present invention can be prepared, for example, by using a transformant into which an expression vector containing a gene sequence encoding the Rh-PDE protein has been introduced. For example, first, this transformant is cultured under appropriate conditions to synthesize the Rh-PDE protein encoded by the gene sequence. Then, the Rh-PDE protein of the present invention can be obtained by recovering the synthesized protein from the transformant or the culture solution.
より具体的に説明すると、適当な発現ベクターに上述のＲｈ−ＰＤＥタンパク質をコードする遺伝子配列を挿入することにより、作製できる。「適当なベクター」とは、原核生物及び真核生物の各種の宿主内で複製保持又は自己増殖できるものであればよく、使用の目的に応じて適宜選択できるものである。その具体例としては、特に限定されず、例えば、特開平１１−２９５９９号公報に記載された公知のベクターが挙げられる。 More specifically, it can be prepared by inserting the gene sequence encoding the above-mentioned Rh-PDE protein into an appropriate expression vector. The “suitable vector” may be any vector as long as it can maintain replication or self-propagation in various prokaryote and eukaryote hosts, and can be appropriately selected depending on the purpose of use. As the specific example, it does not specifically limit, For example, the well-known vector described in Unexamined-Japanese-Patent No. 11-29599 is mentioned.
また、発現ベクターは、培養細胞に導入してタンパク質を調整するのみならず、ヒトやマウス個体に直接導入することで、標的とする生体組織に発現することができる。この場合におけるベクターは、ヒトやマウスに導入可能なベクターを用いる。かかるベクターとしては、アデノ随伴ウイルスベクター（ＡＡＶベクター）、レンチウイルスベクターが好適でありこれらを、例えばマウスの気管支細胞に注射することで導入することができる。 Furthermore, the expression vector can be expressed not only in a cultured cell to prepare a protein, but also directly in a human or mouse individual, thereby expressing it in a target living tissue. The vector in this case is a vector that can be introduced into human or mouse. As such a vector, an adeno-associated virus vector (AAV vector) and a lentiviral vector are suitable and can be introduced, for example, by injection into mouse bronchial cells.
発現ベクターを導入する宿主は、発現ベクターに適合し形質転換され得るものであれば
よく、その具体例としては、特に限定されないが、細菌、酵母、動物細胞、昆虫細胞等の
、公知の天然細胞もしくは人工的に樹立された細胞（特開平１１−２９５９９号公報参照）、あるいは、ヒト、マウス等の動物が挙げられる。形質転換体の培養は、Ｒｈ−ＰＤＥタンパク質が大量にかつ容易に取得できるように、形質転換体の種類等に応じて、公知の栄養培地から適宜選択し、温度、栄養培地のｐＨ、培養時間等を適宜調整して行うことができる（例えば、特開平１１−２９５９９号公報参照）。
The host into which the expression vector is introduced may be any host that is compatible with the expression vector and can be transformed. Specific examples thereof include, but are not limited to, known natural cells such as bacteria, yeast, animal cells, insect cells, etc. Alternatively, artificially established cells (see JP-A-11-29599), or animals such as humans and mice can be mentioned. The culture of the transformant is appropriately selected from known nutrient media according to the type of transformant and the like so that the Rh-PDE protein can be easily obtained in large quantities, and temperature, pH of nutrient medium, culture time And the like can be appropriately adjusted (see, for example, JP-A-11-29599).
Ｒｈ−ＰＤＥタンパク質の単離方法及び精製方法としては、特に限定されず、溶解度を利用する方法、分子量の差を利用する方法、荷電を利用する方法等の公知の方法（例えば、特開平１１−２９５９９号公報参照）が挙げられる。 The method for isolating and purifying the Rh-PDE protein is not particularly limited, and known methods such as a method using solubility, a method using difference in molecular weight, a method using charge, etc. 29599)).
（Ｒｈ−ＰＤＥのアミノ酸配列）
発現されたＲｈ−ＰＤＥの１文字表記のアミノ酸配列（配列番号：１）は以下のようであった。７０４個のアミノ酸が配列したものであった。
(Amino acid sequence of Rh-PDE)
The amino acid sequence of the expressed Rh-PDE in one-letter code (SEQ ID NO: 1) was as follows: 704 amino acids were arranged.
（配列番号：１）
ＭＧＲＫＮＡＡＮＳＳＭＬＱＥＡＳＭＮＮＹＳＭＴＳＡＡＳＧＡＳＳＳＧＲＧＫＲＲＡＫＴＲＮＩＡＩＡＳＴＫＥＶＱＷＱＧＩＦＭＩＩＶＷＬＣＶＭＧＳＬＩＦＦＡＮＰＥＡＳＲＲＶＦＡＫＦＳＨＬＱＳＦＹＧＡＴＳＶＡＦＡＦＡＴＧＬＤＩＬＡＹＶＮＡＶＳＤＥＫＲＶＬＳＧＩＬＡＹＶＤＧＶＡＣＩＳＹＬＳＭＡＴＬＮＬＹＦＬＶＤＳＴＱＧＮＰＶＷＬＭＲＹＡＥＷＩＩＴＣＰＴＬＬＹＷＣＧＬＡＳＲＡＤＲＳＳＶＳＤＩＡＴＡＤＡＬＬＬＡＧＧＡＬＳＳＩＬＰＳＷＰＡＦＦＶＦＡＧＳＦＡＴＹＩＹＶＭＬＨＭＷＧＭＦＧＫＡＭＱＰＤＦＱＰＰＰＰＬＰＲＨＡＬＨＬＬＲＣＥＩＶＭＳＷＳＩＦＰＬＶＥＦＬＲＲＱＧＹＩＤＦＱＶＧＥＡＭＮＣＶＡＤＹＡＡＫＶＧＬＡＭＩＭＶＮＣＮＬＥＱＩＮＡＬＲＶＱＱＭＨＳＡＬＴＧＭＬＫＶＭＲＫＴＮＬＳＳＳＲＭＡＱＬＤＧＶＤＤＤＶＫＳＷＩＭＮＥＦＳＧＳＴＤＧＫＧＧＤＤＡＫＡＱＱＲＡＲＧＲＫＧＨＳＡＡＭＤＡＥＫＬＡＡＳＳＬＦＴＷＤＦＤＡＬＤＫＳＤＥＤＬＴＡＶＣＶＲＶＦＱＥＬＨＶＩＥＱＦＮＩＤＥＫＫＬＲＫＷＬＱＫＶＲＴＱＹＮＫＮＰＦＨＮＷＲＨＡＶＭＶＬＨＴＴＹＬＬＬＴＳＶＡＴＥＦＩＴＥＶＥＬＬＡＭＬＩＡＡＬＳＨＤＬＤＨＮＧＬＴＮＡＦＨＩＮＳＲＳＥＬＡＬＶＹＮＤＱＳＶＬＥＮＨＨＳＨＬＡＦＥＩＬＬＥＫＧＣＮＶＶＥＮＬＤＥＤＥＦＫＲＬＲＡＶＩＩＮＣＩＬＮＴＤＭＡＴＨＨＴＫＬＳＫＭＥＥＶＮＲＬＧＧＦＩＮＬＡＥＮＮＤＱＲＬＦＶＬＡＶＬＬＨＴＡＤＬＨＮＰＩＫＰＦＥＳＮＫＲＷＳＡＲＬＱＫＥＦＮＮＱＶＥＬＥＡＫＭＮＬＰＳＬＰＦＭＲＧＮＤＥＥＳＬＡＫＧＥＩＧＦＩＮＦＶＶＫＰＷＨＱＱＬＳＱＡＦＰＫＬＤＦＬＬＤＴＩＤＡＮＬＡＥＷＫＡＩＡＥＳＹＲＱＭＨ
(SEQ ID NO: 1)
MGRKNAANSSMLQEASMNNYSMTSAASGASSSGRGKRRAKTRNIAIASTKEVQWQGIFMIIVWLCVMGSLIFFANPEASRRVFAKFSHLQSFYGATSVAFAFATGLDILAYVNAVSDEKRVLSGILAYVDGVACISYLSMATLNLYFLVDSTQGNPVWLMRYAEWIITCPTLLYWCGLASRADRSSVSDIATADALLLAGGALSSILPSWPAFFVFAGSFATYIYVMLHMWGMFGKAMQPDFQPPPPLPRHALHLLRCEIVMSWSIFPLVEFLRRQGYIDFQVGEAMNCVADYAAKVGLAMIMVNCNLEQINALRVQQMHSALTGMLKVMRKT LSSSRMAQLDGVDDDVKSWIMNEFSGSTDGKGGDDAKAQQRARGRKGHSAAMDAEKLAASSLFTWDFDALDKSDEDLTAVCVRVFQELHVIEQFNIDEKKLRKWLQKVRTQYNKNPFHNWRHAVMVLHTTYLLLTSVATEFITEVELLAMLIAALSHDLDHNGLTNAFHINSRSELALVYNDQSVLENHHSHLAFEILLEKGCNVVENLDEDEFKRLRAVIINCILNTDMATHHTKLSKMEEVNRLGGFINLAENNDQRLFVLAVLLHTADLHNPIKPFESNKRWSARLQKEFNNQVELEAKMNLPSLPFMRGNDEESLAKGEIGFINFVVK WHQQLSQAFPKLDFLLDTIDANLAEWKAIAESYRQMH
（Ｒｈ−ＰＤＥタンパク質の発現）
Ｓａｌｐｉｎｇｏｅｃａ ｒｏｓｅｔｔａ（ＡＴＣＣ５０８１８株）のゲノム配列中のＲｈ−ＰＤＥをコードする遺伝子配列をＮＣＢＩ Ｇｅｎｅ ＩＤ １６０７８６０６から入手した。
(Expression of Rh-PDE protein)
The gene sequence encoding Rh-PDE in the genomic sequence of Salpingoeca rosetta (strain ATCC 50818) was obtained from NCBI Gene ID 16078606.
入手された遺伝子配列をもとに、ヒトの細胞での発現用に遺伝子配列の最適化をおこない、その遺伝子配列を合成した。合成遺伝子を哺乳類細胞発現用プラスミドであるｐＥＧＦＰ−Ｎ１およびｐｃＤＮＡ３．１にライゲーション法を用いて挿入した。この産物をｐＥＧＦＰ−Ｒｈ−ＰＤＥおよびｐｃＤＮＡ３．１−Ｒｈ−ＰＤＥと呼ぶ。
ｐＥＧＦＰ−Ｒｈ−ＰＤＥおよびｐｃＤＮＡ３．１−Ｒｈ−ＰＤＥをリポフェクション法、又はリン酸カルシウム法によりにＨＥＫ２９３細胞およびＮＤ７／２３細胞に導入し、Ｒｈ−ＰＤＥタンパク質の発現を行った。発現後細胞を回収し、カラムクロマトグラフィー法によりＲｈ−ＰＤＥタンパク質を精製した。
Based on the obtained gene sequence, the gene sequence was optimized for expression in human cells, and the gene sequence was synthesized. The synthetic gene was inserted into plasmids for mammalian cell expression, pEGFP-N1 and pcDNA3.1, using a ligation method. This product is called pEGFP-Rh-PDE and pcDNA3.1-Rh-PDE.
pEGFP-Rh-PDE and pcDNA3.1-Rh-PDE were introduced into HEK293 cells and ND7 / 23 cells by the lipofection method or calcium phosphate method to express Rh-PDE protein. After expression, cells were recovered and Rh-PDE protein was purified by column chromatography.
（Ｒｈ−ＰＤＥの光による活性化メカニズム）
光による活性化メカニズムを調べるために、精製したＲｈ−ＰＤＥタンパク質の光受容部位であるロドプシン部位の光反応特性を分光学的に検証した。試験管内にＲｈ−ＰＤＥを静置して５２０ナノメートルの青緑色の光を照射した。その結果、図１に示すように、Ｒｈ−ＰＤＥは光照射の約７秒後に完全に活性化され、光照射を終えるとその活性は数分かけて減衰し、元の状態に戻ることが分かった。Ｒｈ−ＰＤＥの活性化とは、ｃＡＭＰ、ｃＧＭＰを分解する反応を示すことである。分解産物はそれぞれ５´ＡＭＰ、５´ＧＭＰである。
(Light activation mechanism of Rh-PDE)
In order to investigate the mechanism of activation by light, the light reaction characteristics of the rhodopsin site, which is the light receiving site of the purified Rh-PDE protein, were spectroscopically verified. The Rh-PDE was allowed to stand in a test tube and illuminated with 520 nm blue-green light. As a result, as shown in FIG. 1, it was found that Rh-PDE was fully activated about 7 seconds after light irradiation, and when light irradiation was finished, its activity was attenuated over several minutes and returned to its original state. The The activation of Rh-PDE is to exhibit a reaction for degrading cAMP and cGMP. The degradation products are 5 'AMP and 5' GMP respectively.
試験管内で精製Ｒｈ−ＰＤＥタンパク質とｃＧＭＰを混合し静置して５２０ナノメートルの青緑色の光を、２０分間照射したとき（Ｌｉｇｈｔ）と、しないとき（Ｄａｒｋ）における各時間におけるｃＧＭＰの残存率は、図２のようになった。図２から２０分間の間、ｃＧＭＰの残存率は、両者の場合に共に低下した。光照射をした場合、しない場合よりさらに低下した。 Mixture of purified Rh-PDE protein and cGMP in a test tube and allowed to stand for 520 nm blue-green light for 20 minutes (Light) with and without (Dark) residual rate of cGMP at each time Became like Figure 2. During the period of 20 minutes from FIG. 2, the residual rate of cGMP decreased in both cases. When irradiated with light, it decreased more than the case without.
図２に基づいて、光照射をしたときと光照射をしなかったときのｃＧＭＰの分解量（ｎｍｏｌ／ｍｇ ｔｏｔａｌ ｐｒｏｔｅｉｎ／ｍｉｎ）を算出して図３に示した。図３から、ｃＧＭＰについて、光依存的に１４０％の分解活性の上昇を示すことが判明した。 Based on FIG. 2, the decomposition amount (nmol / mg total protein / min) of cGMP when light irradiation was not performed and light irradiation was calculated is shown in FIG. It was found from FIG. 3 that cGMP showed a 140% increase in degradation activity in a light-dependent manner.
試験管内でＲｈ−ＰＤＥタンパク質とｃＡＭＰを混合し静置して５２０ナノメートルの青緑色の光を、２０分間照射したとき（Ｌｉｇｈｔ）と、しなかったとき（Ｄａｒｋ）における各時間でのｃＡＭＰの残存率は、図４のようになった。図４から２０分間の間、ｃＡＭＰの残存率は、両者の場合に共に低下した。光照射をした場合、しない場合よりさらに低下した。 When mixed with Rh-PDE protein and cAMP in a test tube and allowed to stand and irradiated with 520 nm blue-green light for 20 minutes (Light) and without (Dark) cAMP for each time The survival rate is as shown in FIG. During the period of 20 minutes from FIG. 4, the residual rate of cAMP decreased in both cases. When irradiated with light, it decreased more than the case without.
図４に基づいて、光照射をしたときと光照射をしなかったときのｃＡＭＰの分解量（ｎｍｏｌ／ｍｇ ｔｏｔａｌ ｐｒｏｔｅｉｎ／ｍｉｎ）を算出して図５を示した。図５から、ｃＧＭＰについて、光依存的に１６０％の分解活性の上昇があることが分かった。 Based on FIG. 4, the decomposition amount (nmol / mg total protein / min) of cAMP was calculated with and without light irradiation, and FIG. 5 is shown. From FIG. 5, it was found that for cGMP, there is a 160% increase in degradation activity in a light-dependent manner.
（ＮＤ７／２３細胞へのＲｈ−ＰＤＥの導入）
上述した哺乳類細胞発現用プラスミドであるｐＥＧＦＰ−Ｒｈ−ＰＤＥを、リポフェクション法によりマウス由来の培養細胞に導入した。なお、ｐＥＧＦＰ−Ｒｈ−ＰＤＥには緑色蛍光タンパク質（ＧＦＰ）遺伝子も含まれており、Ｒｈ−ＰＤＥタンパク質の発現を緑色蛍光により検出することができる。遺伝子導入後の細胞を、蛍光顕微鏡を用いて観察したところ細胞内に緑色蛍光が観察されたため（図６（ｂ））、Ｒｈ−ＰＤＥが発現したと結論付けた。
(Introduction of Rh-PDE into ND7 / 23 cells)
The above-described mammalian cell expression plasmid pEGFP-Rh-PDE was introduced into cultured cells of mouse origin by the lipofection method. In addition, green fluorescence protein (GFP) gene is also contained in pEGFP-Rh-PDE, and the expression of Rh-PDE protein can be detected by green fluorescence. It was concluded that Rh-PDE was expressed because green fluorescence was observed in the cells when the cells after gene transfer were observed using a fluorescence microscope (FIG. 6 (b)).
（ＨＥＫ２９３細胞へのＲｈ−ＰＤＥの導入）
ヒトの細胞の中でも同じようにＲｈ−ＰＤＥが働くかを検証するために、Ｒｈ−ＰＤＥをヒトの培養細胞での一種であるＨＥＫ２９３細胞に導入した。このために、ｐＥＧＦＰ−Ｒｈ−ＰＤＥをリポフェクション法を用いて導入した。
(Introduction of Rh-PDE into HEK 293 cells)
In order to verify whether Rh-PDE works in human cells as well, Rh-PDE was introduced into HEK293 cells, which is a kind of cultured human cells. For this, pEGFP-Rh-PDE was introduced using the lipofection method.
（ｃＡＭＰ濃度の経時的検出）
Ｒｈ−ＰＤＥを導入したＨＥＫ２９３細胞に青緑光（５１０ナノメートル）を照射して、細胞内のｃＡＭＰ濃度を経時的に検出することで、青緑色光照射に応じたｃＡＭＰ濃度の減少を観察した。
(Detection of cAMP concentration over time)
By irradiating blue-green light (510 nm) to the HEK 293 cells into which Rh-PDE has been introduced and detecting intracellular cAMP concentration over time, a decrease in cAMP concentration in response to blue-green light irradiation was observed.
図７に示すように、２分間青緑光を照射すると、細胞内ｃＡＭＰ量を示す相対ルミネセンスは一旦減少し、その後漸増して約６分後には減少前の値に戻った。減少前の値は、青緑光を照射しなかったときのｃＡＭＰの相対ルミネセンスであるから、Ｒｈ−ＰＤＥは人の細胞内に導入されても、試験管内と同様な光による活性化メカニズムを有することが判明した。
なお、図７中のＡＴＲはａｌｌ−ｔｒａｎｓ−ｒｅｔｉｎａｌ（全トランスレチナール）の略称であって、ビタミンＡの誘導体でヒトの生体内にも存在するが、ｃＡＭＰの相対ルミネセンスの感度を向上させるため、添加されたものである。図７において＋ＡＴＲのときが−ＡＴＲのときよりもｃＡＭＰの相対ルミネセンスが減少していた。
As shown in FIG. 7, when blue-green light was applied for 2 minutes, the relative luminescence indicating the amount of intracellular cAMP once decreased, then gradually increased and returned to the value before the decrease after about 6 minutes. Since the value before reduction is the relative luminescence of cAMP when not irradiated with blue-green light, Rh-PDE, even when introduced into human cells, has an activation mechanism by light similar to that in vitro. It has been found.
Note that ATR in FIG. 7 is an abbreviation of all-trans-retinal (all-trans retinal), which is a derivative of vitamin A and is also present in human beings, but in order to improve the sensitivity of relative luminescence of cAMP. , Was added. In FIG. 7, the relative luminescence of cAMP was reduced when + ATR was -ATR.
図７の青緑光照射を１１回繰り返した結果を図８に示す。図８から、図７のｃＡＭＰの相対ルミネセンスの挙動が１１回繰り返されていたため、ＨＥＫ２９３細胞に導入されたＲｈ−ＰＤＥについて、可逆的、繰り返しの光操作が可能であることが判明した。 The result of repeating the blue-green light irradiation of FIG. 7 11 times is shown in FIG. From FIG. 8, it was found that reversible and repetitive light manipulation was possible for Rh-PDE introduced into HEK 293 cells because the relative luminescence behavior of cAMP in FIG. 7 was repeated 11 times.
細胞内環状ヌクレオチド濃度の光操作がＲｈ−ＰＤＥを用いることで可能であり、Ｒｈ−ＰＤＥを応用した細胞内シグナル伝達の光操作法を確立する。これにより環状ヌクレオチドが関わる疾患メカニズムの解明に貢献する。環状ヌクレオチドの欠乏は気管支喘息、アルツハイマー病、網膜色素変性症等の原因である。Ｒｈ−ＰＤＥを健常な動物組織に導入することで、環状ヌクレオチド濃度を光照射により低下させることが可能になるため、疾患状態を迅速に作り出すことが可能となる。これは疾患メカニズムの解明、さらには治療法の開発に貢献する画期的な手法となる。 The light manipulation of intracellular cyclic nucleotide concentration is possible by using Rh-PDE, and a light manipulation method of intracellular signaling by applying Rh-PDE is established. This contributes to the elucidation of the disease mechanism in which cyclic nucleotides are involved. Depletion of cyclic nucleotides is the cause of bronchial asthma, Alzheimer's disease, retinitis pigmentosa and the like. By introducing Rh-PDE into healthy animal tissue, it is possible to reduce the cyclic nucleotide concentration by light irradiation, and thus it is possible to rapidly create a disease state. This is an innovative method that contributes to the elucidation of disease mechanisms and the development of therapeutics.
Claims (2)
A rhodopsin phosphodiesterase consisting of an amino acid in which one or more amino acids are deleted, substituted or added in claim 1 and having cGMP and / or cAMP degradation activity by light irradiation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2017174601A JP2019047763A (en) | 2017-09-12 | 2017-09-12 | Rhodopsin phosphodiesterase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2017174601A JP2019047763A (en) | 2017-09-12 | 2017-09-12 | Rhodopsin phosphodiesterase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2019047763A true JP2019047763A (en) | 2019-03-28 |
Family
ID=65904660
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2017174601A Pending JP2019047763A (en) | 2017-09-12 | 2017-09-12 | Rhodopsin phosphodiesterase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2019047763A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008505193A (en) * | 2004-06-10 | 2008-02-21 | ジェネンコア・インターナショナル，インコーポレイテッド | Compositions containing various proteorhodopsins and / or bacteriorhodopsins and uses thereof |
| JP2009502140A (en) * | 2005-07-22 | 2009-01-29 | ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー | Photoactivated cation channel and use thereof |
-
2017
- 2017-09-12 JP JP2017174601A patent/JP2019047763A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008505193A (en) * | 2004-06-10 | 2008-02-21 | ジェネンコア・インターナショナル，インコーポレイテッド | Compositions containing various proteorhodopsins and / or bacteriorhodopsins and uses thereof |
| JP2009502140A (en) * | 2005-07-22 | 2009-01-29 | ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー | Photoactivated cation channel and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| NUCLEIC ACIDS RESEARCH, vol. vol 10, Number 20, JPN6020038113, 1982, pages 6487 - 6500, ISSN: 0004791593 * |
| UNIPROT [ONLINE], vol. F2TZN0 version 36, JPN6021031969, 7 June 2017 (2017-06-07), pages 2021 - 8, ISSN: 0004791592 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11446398B2 (en) | Regulated biocircuit systems | |
| Takahashi et al. | AUREOCHROME, a photoreceptor required for photomorphogenesis in stramenopiles | |
| US11879145B2 (en) | Reagents and methods for replication, transcription, and translation in semi-synthetic organisms | |
| KR20170121291A (en) | Engineered bacteria to treat diseases that benefit from reduced intestinal inflammation and / or enhanced intestinal mucosal barriers | |
| CN109803977A (en) | Nucleic acid product and its method of administration | |
| WO2002072846A9 (en) | Synthetic genes and bacterial plasmids devoid of cpg | |
| CN107849574A (en) | Increase the UTR of the translation efficiency of RNA molecule | |
| US20220243244A1 (en) | Compositions and methods for in vivo synthesis of unnatural polypeptides | |
| CN101586155A (en) | Method for diagnosing non-small cell lung cancers | |
| US20190284164A1 (en) | Photocleavable Chemical Inducers of Dimerization (CID) and Methods of Use | |
| Adin et al. | Mutations in ampG and lytic transglycosylase genes affect the net release of peptidoglycan monomers from Vibrio fischeri | |
| Zhang et al. | Holomycin, an antibiotic secondary metabolite, is required for biofilm formation by the native producer Photobacterium galatheae S2753 | |
| Pan et al. | Screening and identification of the host proteins interacting with Toxoplasma gondii rhoptry protein ROP16 | |
| Sabui et al. | Identification of transmembrane protein 237 as a novel interactor with the intestinal riboflavin transporter-3 (RFVT-3): role in functionality and cell biology | |
| Pak et al. | Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo | |
| Csonka et al. | Biosynthesis of proline | |
| JP2019047763A (en) | Rhodopsin phosphodiesterase | |
| Henriques et al. | Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome | |
| KR101898178B1 (en) | Biosensor for detecting glutamate and preparation method thereof | |
| Boas Lichty et al. | NhaR, LeuO, and H-NS Are Part of an Expanded Regulatory Network for Ectoine Biosynthesis Expression | |
| Tsogbadrakh et al. | Expression of Anabaena sensory rhodopsin is influenced by different codons of seven residues at the N-terminal region | |
| CA2533479C (en) | Peptide protein translation inhibitor and the use thereof for protein translation control | |
| Kumar | Differential Activations of Dead Box RNA Helicases RhlB and Rhie By Hfq-sRNA and Their Target mRNAs | |
| Zhang et al. | Multiomics integration for the function of bacterial outer membrane vesicles in the larval settlement of marine sponges | |
| Misic | Regulation of mtDNA gene expression and the role of respiratory chain supercomplexes in mammalian mitochondria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A80 | Written request to apply exceptions to lack of novelty of invention |
Free format text: JAPANESE INTERMEDIATE CODE: A80 Effective date: 20171006 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20200901 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20210824 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20211005 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20211102 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20211216 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220221 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20220222 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20220607 |