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: 10683396
• 负责人: Shuichi Hoshika
• 金额: $ 15.37万
• 依托单位: FOUNDATION FOR APPLIED MOLECULAR EVOLUTN
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/10683396
• 关键词: Amino Acids; Aminoacylation; Antibodies; Anticodon; Antineoplastic Agents; Automobile Driving; Behavior; Biological Process; Cancer Biology; Cells; Charge; Codon Nucleotides; Complement; Complex; DNA; Development; Drug Screening; Drug Targeting; Elongation Factor; Engineering; Enzymes; Epigenetic Process; Escherichia coli; Eukaryotic Cell; Foundations; Hand; Hydrogen Bonding; Immunoassay; In Vitro; Information Systems; JAK2 gene; Japanese; Letters; Ligase; Lysine; Malignant Neoplasms; Measures; Modernization; Modification; Molecular; Molecular Evolution; Nucleotides; Outcome; Performance; Pharmaceutical Preparations; Phenotype; Phosphoserine; Phosphotyrosine; Positioning Attribute; Post-Translational Protein Processing; Posttranslational Amino Acid Modification; Procedures; Protein Engineering; Proteins; Proteomics; Purines; Pyrimidines; RNA; Research; Research Personnel; Ribosomes; Risk; Services; Site; Specific qualifier value; Speed; Standardization; System; Technology; Thermodynamics; Time; Transfer RNA; Transfer RNA Aminoacylation; Translations; Tyrosine; United States National Institutes of Health; Vision; Work; Writing; anticancer research; cancer cell; cancer proteomics; clinically relevant; drug related cancer; experimental study; genetic information; improved; inhibitor; prohibitin; protein protein interaction; success; synthetic biology; therapeutic protein; tool

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.

高质量的蛋白质具有多次翻译后修改 应用分子进化基础 Shuichi Hoshika 抽象的 拟议的技术将通过体外翻译（IVT）来制造持有非典型氨基的蛋白质 酸通常仅通过翻译后修饰（PTM）放入。立即交付将是技术 - 具有三个PTM-AAS（乙酰透析，磷酸磷酸，磷酸酪氨酸）的蛋白质的蛋白质 在许多情况下，长期（使用300-1100个氨基酸蛋白）蛋白质的精确位置占95％。 NCI本身通过呼吁制造这种蛋白质的工具来激发该提议，癌症研究人员 整个癌症蛋白质组学的需求。今天，这种蛋白质仅通过与生活真核隔离一起使用 细胞。这些很少是纯净的。我们的纯PTM蛋白将用于获取抗体，识别PTM特征 癌症，将定量免疫测定标准化为标准，发现与癌症有关的抑制剂和药物 其与药物相关的形式的酶，并研究蛋白质 - 蛋白质相互作用。这些将迅速获得 并且在自己的实验室（带有体外翻译套件）或通过服务公司（与抗体有关）的实验室中廉价。 作为绩效指标，NCI定义为“可用量”为“ 0.5至1 mg蛋白质”，“ 50-- 在指定站点上进行80％的修改”。我们的技术会做得更好，从而产生1-10毫克的蛋白质 在许多指定地点，三种不同的PTM氨基酸的修饰> 95％。 该项目的背后是DNA和RNA（XNA）的合成生物学的持续革命 扩展的XNA，PI在2019年增强。人工XNA看起来像标准XNA。但是，它添加了对 洗牌氢键基团，使扩展的XNA在蛋白质“词典”中“写”更多的aa“单词”。 与IMAT R21格式一致，该项目将证明概念，脱离风险程序，并采用足够的概念 指导NCI试图完成这种变革技术的步骤。我们将仅使用8个“ Hachimoji” 核苷酸以三个目的是系统地添加这些PTM-AAS，同时开发了控制概念以控制 必须控制以应对这一巨大挑战的互动：（i）在hachimoji密码子和反元中 在翻译过程中，（ii）在氨基酰化过程中合成酶和hachimoji反激代物之间的（iii）之间（iii）之间（ii） 正交Hachimoji充满了大肠杆菌核糖体复合物的TRNA和一部分。即使范围有限， 由于这些PTM-AAS的重要性，该技术将具有变革性。满足了目标，我们的 通过制作禁止素2（这3个PTM-AAS的7个示例），将显示出达到绩效指标的能力 使用增强的IVT（EIVT）。 作为长期交付 使用该技术的蛋白质，一种更具变革性的结果。接下来，随着研究人员的进步和移动 在长期视力中，Hachimoji DNA进入活细胞，更具变革性的结果是可能的。