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：
Shuichi Hoshika
金额：
$ 15.37万
依托单位：
FOUNDATION FOR APPLIED MOLECULAR EVOLUTN
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：
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

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.

具有多种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抽象的拟议的技术将通过体外翻译（IVT）制造含有非规范氨基的蛋白质通常仅通过翻译后修饰（PTM）添加酸。提供含有三种 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蛋白质（使用 300 - 1100 个氨基酸蛋白质）的许多精确位置中，占据率 >95%。 NCI 本身通过呼吁开发制造此类蛋白质的工具来推动这一提议，癌症研究人员可以利用这些工具如今，此类蛋白质只能通过从活体真核生物中分离来获得。这些细胞很少是纯的。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，发现癌症相关的抑制剂和药物酶及其药物相关形式，并研究蛋白质-蛋白质相互作用，这些将很快获得。并且可以在自己的实验室（使用体外翻译套件）或通过服务公司（对于抗体）廉价地进行。作为绩效衡量标准，NCI 将“可用量”定义为“0.5 至 1 毫克蛋白质”，其中“50- 在指定位点进行 80% 的修饰”。我们的技术会做得更好，产生 1-10 毫克的蛋白质在许多指定位点对三种不同的 PTM 氨基酸进行 >95% 的修饰。该项目背后是 DNA 和 RNA (xNA) 合成生物学领域正在进行的革命，它带来了扩展的 xNA，由 PI 于 2019 年增强，看起来像标准 xNA，但它增加了对。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、消除风险程序并采取足够的措施指导 NCI 寻求完成这项变革性技术的步骤我们将仅使用 8 个“hachimoji”。核苷酸在三个目标中系统地添加这些 PTM-AA，同时开发控制这些 PTM-AA 的概念为了应对这一重大挑战，必须控制相互作用：（i）八字密码子和反密码子之间的相互作用翻译过程中，(ii) 氨酰化过程中合成酶和八字反密码子之间，以及 (iii) 之间正交 hachimoji 带电 tRNA 和大肠杆菌核糖体复合物的一部分即使范围有限，由于这些 PTM-AA 的重要性，当我们的目标得到实现时，该技术将具有变革性。满足绩效衡量标准的能力将通过制作 Prohibitin 2（这 3 个 PTM-AA 的 7 个示例）来显示使用增强型 IVT (eIVT)。作为长期交付，由于 8 字母 DNA 可以交付 512 个密码子，所有 PTM AA 都可以纳入接下来，随着研究人员推进 IVT 和移动，使用该技术的蛋白质将产生更具变革性的成果。将八字 DNA 转化为活细胞，从长远来看，甚至可能产生更多变革性的结果。