WO2020238433A1 - Construction d'un système d'aminoacyl-arnt synthétase/arnt orthogonal à l'aide d'un procédé de conception chimérique - Google Patents

Construction d'un système d'aminoacyl-arnt synthétase/arnt orthogonal à l'aide d'un procédé de conception chimérique Download PDF

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WO2020238433A1
WO2020238433A1 PCT/CN2020/084079 CN2020084079W WO2020238433A1 WO 2020238433 A1 WO2020238433 A1 WO 2020238433A1 CN 2020084079 W CN2020084079 W CN 2020084079W WO 2020238433 A1 WO2020238433 A1 WO 2020238433A1
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trna
chimeric
trna synthetase
aminoacyl
synthetase
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PCT/CN2020/084079
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Chinese (zh)
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林世贤
柳光龙
丁文龙
赵红霞
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浙江大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • Protein is the main substance that functions in the organism. 20 amino acids encoded by 61 codons are synthesized in the ribosome. Although the 20 kinds of amino acids endow proteins with the characteristics of participating in various physiological and biochemical activities, only a few active groups such as sulfhydryl and hydroxyl groups can be chemically manipulated. In order to better study the physiological functions of proteins, the genetic code expansion technology that can introduce other unnatural amino acids with active groups came into being.
  • the genetic code expansion technology uses an orthogonalized aminoacyl tRNA synthetase/tRNA system to identify unnatural amino acids with different functions and decode unallocated codons (stop codons, quadruple codons, etc.) to achieve site-directed insertion of unnatural amino acids. So far, this system has achieved the insertion of more than 150 unnatural amino acids with different active groups, performing different functions, such as biological tracking and imaging, regulation of protein functions in the body, research on post-translational modifications, and proteomics Analysis and biological treatment.
  • S5 For the low-efficiency chimeric system, select the acceptor arm of the chimeric tRNA and construct a mutation library for screening.
  • S7 Construction of chimeric aminoacyl-tRNA synthetase vector.
  • S12 Synthesize connecting peptides of different lengths and types and load them between the two parts of the chimeric aminoacyl-tRNA synthetase.
  • Types of connecting peptides GS-rich, helix, P-rich, the sequence is shown in Table 3-1.
  • Figure 9 shows the chimeric histidine system.
  • A Coomassie brilliant blue staining analysis of purified GFP protein with histidine inserted
  • B Mass spectrometry analysis to confirm the insertion of histidine in GFP
  • C LC-MS/MS Analysis confirms the insertion of histidine in GFP
  • D non-denaturing polyacrylamide gel electrophoresis analysis of histidine insertion of GFP protein
  • E chimeric histidyl-tRNA/tRNA system with different histidines at 22°C or 30°C Amber suppression efficiency of acid concentration.
  • Figure 11 shows the optimization of chimeric histidine tRNA synthetase.
  • Figure A Overview of chHisRS linked peptides of different lengths; B: GFP reporter method and native gel fluorescence analysis of chHisRS amber inhibitory activity of different lengths; C: The GFP reporter method analyzes the amber inhibitory activity of chHisRS carrying different types of connecting peptides.
  • GS rich means that the connecting peptides are rich in Gly and Ser
  • Pro rich means that the connecting peptides are rich in Pro
  • the helix means that the connecting peptide is a helical structure.
  • the lysate was centrifuged at 12,000 rpm and 4°C for 60 minutes, and the supernatant obtained was loaded to the nickel affinity chromatography chelating chromatography that was equilibrated with NTA-0 buffer in advance, and then washed with 6 times volume of NTA-0 buffer containing 50 mM imidazole . Finally, the protein was eluted with NTA-0 buffer added with 500 mM imidazole. The purified protein was analyzed by SDS-PAGE and LC-MS.
  • the purified protein was analyzed on an LCQ Deca XP MAX mass spectrometer (Thermo Fisher Science) equipped with an electrospray ionization (ESI) source and an Agilent 1200 HPLC. Separation and desalination were carried out on Agilent 300SB-C18 column (300 ⁇ 2.1, 150mm, 5 ⁇ m).
  • the mobile phase A was set to an aqueous solution containing 0.1% formic acid, and the mobile phase B contained 0.1% formic acid in acetonitrile, and the flow rate was set to 0.200 ml/min.
  • Use XCalbur-Quar browser software to analyze the data.
  • the chimeric seryl-tRNA synthetase/tRNA system constructed by the above examples has low activity.
  • the receptor arm region of the chimeric serine tRNA is modified, and the specific steps are as follows:
  • Example 8 Analysis of amber inhibition efficiency of chimeric histidyl-tRNA synthetase/tRNA system in mammalian cells

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Wood Science & Technology (AREA)
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  • Physics & Mathematics (AREA)
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  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne la construction d'un système d'aminoacyl-ARNt synthétase/ARNt orthogonal en utilisant un procédé de conception chimérique. Le système greffe l'orthogonalité universelle d'eucaryotes et de procaryotes d'un système pyrrolysyl-ARNt synthétase/ARNt au moyen d'une conception chimérique rationnelle, comprend plusieurs systèmes d'ARNt synthétase/ARNt chimériques, tels que l'histidine, la phénylalanine, l'alanine et les systèmes de sérine, et peut être appliqué à des procaryotes et à des eucaryotes, ainsi qu'à la transformation orthogonale d'autres paires aminoacyl-ARNt synthétase/ARNt.
PCT/CN2020/084079 2019-05-24 2020-04-10 Construction d'un système d'aminoacyl-arnt synthétase/arnt orthogonal à l'aide d'un procédé de conception chimérique WO2020238433A1 (fr)

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CN201910440254.8 2019-05-24
CN201910440254.8A CN110172467B (zh) 2019-05-24 2019-05-24 一种利用嵌合设计方法构建正交的氨酰-tRNA合成酶/tRNA体系

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CN110172467B (zh) * 2019-05-24 2021-03-16 浙江大学 一种利用嵌合设计方法构建正交的氨酰-tRNA合成酶/tRNA体系
CN114026232A (zh) * 2019-06-21 2022-02-08 波士顿学院董事会 哺乳动物细胞中非天然氨基酸掺入的增强平台
CN111118048B (zh) * 2019-11-11 2022-06-10 浙江大学 嵌合苯丙氨酰-tRNA合成酶/tRNA的应用
CN114250243B (zh) * 2020-09-24 2024-05-03 深圳华大生命科学研究院 一种检测极端嗜盐生物中氨酰-tRNA合成酶和tRNA活性的系统及方法
CN114134141B (zh) * 2021-11-03 2024-01-30 杭州嵌化合生医药科技有限公司 一种引入非天然氨基酸的嵌合体苯丙氨酸翻译系统及其构建方法
CN114940979B (zh) * 2022-02-16 2024-01-23 杭州嵌化合生医药科技有限公司 一种利用遗传密码扩展提高阳离子-π相互作用的方法及应用
CN114657156A (zh) * 2022-03-11 2022-06-24 南京中医药大学 吡咯赖氨酸氨酰-tRNA合成酶突变体及其筛选系统和应用
CN114908066B (zh) * 2022-05-17 2024-01-23 杭州嵌化合生医药科技有限公司 一种正交翻译系统及其在再分配密码子恢复ptc疾病中功能蛋白表达方面的应用

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