High Quality Proteins with Multiple Post Translational Modifications
具有多种翻译后修饰的高质量蛋白质
基本信息
- 批准号:10683396
- 负责人:
- 金额:$ 15.37万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-09-22 至 2024-08-31
- 项目状态:已结题
- 来源:
- 关键词:Amino AcidsAminoacylationAntibodiesAnticodonAntineoplastic AgentsAutomobile DrivingBehaviorBiological ProcessCancer BiologyCellsChargeCodon NucleotidesComplementComplexDNADevelopmentDrug ScreeningDrug TargetingElongation FactorEngineeringEnzymesEpigenetic ProcessEscherichia coliEukaryotic CellFoundationsHandHydrogen BondingImmunoassayIn VitroInformation SystemsJAK2 geneJapaneseLettersLigaseLysineMalignant NeoplasmsMeasuresModernizationModificationMolecularMolecular EvolutionNucleotidesOutcomePerformancePharmaceutical PreparationsPhenotypePhosphoserinePhosphotyrosinePositioning AttributePost-Translational Protein ProcessingPosttranslational Amino Acid ModificationProceduresProtein EngineeringProteinsProteomicsPurinesPyrimidinesRNAResearchResearch PersonnelRibosomesRiskServicesSiteSpecific qualifier valueSpeedStandardizationSystemTechnologyThermodynamicsTimeTransfer RNATransfer RNA AminoacylationTranslationsTyrosineUnited States National Institutes of HealthVisionWorkWritinganticancer researchcancer cellcancer proteomicsclinically relevantdrug related cancerexperimental studygenetic informationimprovedinhibitorprohibitinprotein protein interactionsuccesssynthetic biologytherapeutic proteintool
项目摘要
High Quality Proteins with Multiple Post Translational Modifications
Foundation for Applied Molecular Evolution
Shuichi Hoshika
ABSTRACT
The proposed technology will make, by in vitro translation (IVT), proteins that hold non-canonical amino
acids normally put in only by post-translational modification (PTM). The immediate deliverable will be tech-
nology that delivers proteins with three PTM-AAs (acetyllysine, phosphserine, phosphotyrosine) incorporated
in many, exact positions in long (300 - 1100 amino acid proteins are used) proteins with >95% occupancy.
The NCI itself motivated this proposal by its calls for tools to make such proteins, which cancer researchers
need throughout cancer proteomics. Today, such proteins are available only via isolation from living eukaryotic
cells. These are rarely pure. Our pure PTM proteins will be used to get antibodies, identify PTM signatures of
cancers, standardize quantitative immunoassays as standards, discover inhibitors and drugs for cancer-related
enzymes in their drug-relevant forms, and study protein-protein interactions. These will be obtained rapidly
and inexpensively in their own labs (with in vitro translation kits) or via service companies (as for antibodies).
As Performance Measures, the NCI defined "useable amounts" to be "0.5 to 1 mg of protein" with "50-
80% modification at the specified site". Our technology will do better, generating 1-10 mg of protein with
>95% modification for three different PTM amino acids at many specified sites.
Behind this project is an ongoing revolution in the synthetic biology of DNA and RNA (xNA) that delivered
expanded xNA, enhanced in 2019 by the PI. Artificial xNA looks like standard xNA. However, it adds pairs by
shuffling hydrogen bonding groups, allowing expanded xNA to “write” more AA “words” in a protein “lexicon”.
Consistent with an IMAT R21 format, this project will prove concepts, de-risk procedures, and take enough
steps to guide the NCI as it seeks to complete this transformative technology. We will use only 8 "hachimoji"
nucleotides to systematically add, in three Aims, these PTM-AAs while developing the concepts to control the
interactions that must be controlled to meet this grand challenge: (i) between hachimoji codons and anticodons
during translation, (ii) between synthetases and hachimoji anticodons during aminoacylation, and (iii) between
orthogonal hachimoji charged tRNAs and parts of the E. coli ribosome complex. Even with this limited scope,
the technology will be transformative because of the importance of these PTM-AAs. As Aims are met, our
ability to meet Performance Measures will be shown by making Prohibitin 2 (7 exemplars of these 3 PTM-AAs)
using enhanced IVT (eIVT) .
As a long term deliverable, since 8-letter DNA can deliver 512 codons, all PTM AAs can be incorporated into
proteins using this technology, a more transformative outcome. Next, as researchers advance IVT and move
hachimoji DNA into living cells, even more transformative outcomes are possible in a long term vision.
具有多个翻译后修饰的高质量蛋白质
项目成果
期刊论文数量(0)
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Shuichi Hoshika其他文献
Shuichi Hoshika的其他文献
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{{ truncateString('Shuichi Hoshika', 18)}}的其他基金
High Quality Proteins with Multiple Post Translational Modifications
具有多种翻译后修饰的高质量蛋白质
- 批准号:
10271814 - 财政年份:2021
- 资助金额:
$ 15.37万 - 项目类别:
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