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
• 负责人: Shuichi Hoshika
• 金额: $ 15.68万
• 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/10271814
• 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 Population; Letters; Ligase; 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; base; cancer cell; cancer proteomics; clinically relevant; drug related cancer; experimental study; genetic information; improved; inhibitor/antagonist; 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.

具有多个翻译后修饰的高质量蛋白质 应用分子进化基金会 星香秀一 摘要 这项技术将通过体外翻译（IVT），使蛋白质含有非规范的氨基。 通常仅通过翻译后修饰（PTM）加入。立即交付的将是技术- 递送掺入三种PTM-AA（乙酰赖氨酸、磷酸丝氨酸、磷酸酪氨酸）的蛋白质的技术 在长（使用300 - 1100个氨基酸的蛋白质）蛋白质中的许多精确位置具有>95%的占有率。 NCI本身通过呼吁制造这种蛋白质的工具来推动这一提议，癌症研究人员 癌症蛋白质组学的需求。今天，这种蛋白质只能通过从活的真核生物中分离出来 细胞它们很少是纯的。我们的纯PTM蛋白将用于获得抗体，鉴定PTM特征， 癌症，标准化定量免疫测定作为标准，发现癌症相关的抑制剂和药物 研究药物相关形式的酶，并研究蛋白质-蛋白质相互作用。这些将很快得到 并且在他们自己的实验室（使用体外翻译试剂盒）或通过服务公司（如抗体）便宜地进行。 作为性能指标，NCI将“可用量”定义为“0.5至1 mg蛋白质”，“50- 100 mg蛋白质”。 80%的修改在指定的网站”。我们的技术将做得更好，产生1-10毫克的蛋白质， 在许多特定位点，三种不同PTM氨基酸的修饰>95%。 在这个项目的背后，是DNA和RNA（xNA）合成生物学的一场持续革命， 扩展的xNA，在2019年由PI增强。人工xNA看起来像标准xNA。但是，它通过以下方式添加对： 改组氢键基团，允许扩展的xNA在蛋白质“词典”中“写入”更多AA“单词”。 与IMAT R21格式一致，该项目将证明概念，去风险程序，并采取足够的 在NCI寻求完成这一变革性技术时，指导其采取步骤。我们只用8个“八王子” 核苷酸系统地添加，在三个目标，这些PTM-AA，同时发展的概念，以控制 必须控制的相互作用，以满足这一巨大的挑战：（i）在八王子密码子和反密码子之间 在翻译过程中，（ii）在氨酰化过程中，合成酶和hachimoji反密码子之间，和（iii） 正交hachimoji带电荷的tRNA和E.大肠杆菌核糖体复合物。即使在这个有限的范围内， 由于这些PTM-AA的重要性，该技术将是变革性的。随着目标的实现，我们 通过制定禁止2（这3个PTM-AA的7个示例）来证明满足性能指标的能力 使用增强型IVT（eIVT）。 作为长期可递送物，由于8个字母的DNA可以递送512个密码子，因此所有PTM AA都可以并入到 使用这项技术的蛋白质，一个更具变革性的成果。接下来，随着研究人员推进IVT， 将八王子的DNA转化为活细胞，从长远来看，甚至可能产生更多的变革性成果。