High Quality Proteins with Multiple Post Translational Modifications

具有多个翻译后修饰的高质量蛋白质 应用分子进化基础 星间水一 摘要 这项拟议的技术将通过体外翻译(IVT)来制造含有非典型氨基的蛋白质。 酸通常只有通过翻译后修饰(PTM)才能加入。眼前的成果将是科技- 提供含有三种PTM-AAs(乙酰赖氨酸、磷酸丝氨酸、磷酸酪氨酸)的蛋白质的Nology 在许多情况下，长蛋白质(使用300-1100个氨基酸的蛋白质)中的准确位置占据了95%的位置。 NCI本身是通过呼吁使用工具来制造这种蛋白质来推动这一提议的，癌症研究人员 需要贯穿于癌症蛋白质组学。今天，这种蛋白质只能通过从活的真核生物中分离得到。 细胞。这些东西很少是纯净的。我们的纯PTM蛋白将用于获得抗体，鉴定PTM信号 癌症，标准化定量免疫分析作为标准，发现癌症相关的抑制剂和药物 酶的药物相关形式，并研究蛋白质-蛋白质相互作用。这些将很快获得 并廉价地在自己的实验室(使用体外翻译试剂盒)或通过服务公司(如抗体)。 作为绩效衡量标准，NCI将“可使用量”定义为“0.5至1毫克蛋白质”，其中“50- 我们的技术会做得更好，可以产生1-10毫克的蛋白质 &gt；三种不同的PTM氨基酸在多个指定位点的95%修饰。 在这个项目的背后是DNA和RNA合成生物学(XNA)正在进行的革命，它提供了 扩展的XNA，2019年由PI增强。人造XNA看起来像标准的XNA。但是，它通过以下方式添加对 重组氢键基团，使扩展后的XNA能够在蛋白质词典中写出更多的AA单词。 与IMAT R21格式一致，该项目将验证概念、降低风险的程序，并采取足够的 当NCI寻求完成这项变革性技术时，指导它的步骤。只用8个“八条鱼”。 核苷酸在三个目标中系统地添加这些PTM-AA，同时开发概念来控制 为了迎接这一重大挑战必须控制的相互作用：(I)八字密码和反密码子之间的相互作用 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，以及(Iii)在 正交hachimoji带电的tRNA和部分大肠杆菌核糖体复合体。即使在这个有限的范围内， 由于这些PTM-AA的重要性，这项技术将具有变革性。随着目标的实现，我们的 将通过制造Prohibitin 2(这3个PTM-AA中的7个样本)来显示满足绩效衡量标准的能力 使用增强型IVT(EIVT)。 作为一种长期交付，由于8个字母的DNA可以提供512个密码子，所有的PTM AA都可以合并到 使用这项技术的蛋白质，结果更具变革性。接下来，随着研究人员推进IVT并 将hachimoji DNA注入活细胞，从长远来看，更具变革性的结果是可能的。

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.