The GCE4All Center: Unleashing the Potential of Genetic Code Expansion for Biomedical Research

GCE4All 中心：释放遗传密码扩展在生物医学研究中的潜力

• 批准号: 10799462
• 负责人: RYAN A MEHL
• 金额: $ 25万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10799462
• 关键词: Acceleration; Amino Acids; Biological Process; Biomedical Research; Biomedical Technology; Cancer Etiology; Development; Diabetes Mellitus; Diagnostic tests; Disease; Education; Educational workshop; Equipment; Funding; Genetic Code; Grant; Heart Diseases; Human Resources; Label; Life; Longevity; Maintenance; Mission; Oregon; Pain; Parkinson Disease; Persons; Pharmaceutical Preparations; Process; Proteins; Reaction; Research; Research Personnel; Running; Services; Site; Source; System; Technology; Training; Translations; Universities; Visualization; Walking; Water; Work; cellular engineering; design; innovation; instrument; member; repository; technology development; tool

EQUIPMENT SUPPLEMENT ABSTRACT The GCE4All Biomedical Technology Development and Dissemination Center at Oregon State University (OSU) serves to optimize, develop, and broadly disseminate Genetic Code Expansion (GCE) technology – the engineering of cellular translation to express proteins containing non-canonical amino acids (ncAAs). During its envisioned lifespan of ≤15 years, the Center's mission is to effectively extend existing GCE technologies for facile use by non-specialists, and to broadly disseminate them via widespread education, effective training, and by providing sustainable access to optimized technologies via established repositories – enabling powerful GCE approaches to become standard, widely-used tools of biomedical researchers. As we build new GCE encoding systems, quantifying the intact mass of the ncAA-proteins is critical to verify the site-specific encoding of ncAAs, to verify that there is no loss in fidelity by mis-encoding of natural amino acids, and to evaluate biorthogonal labeling reactions on proteins. However, the center does not have equipment to characterize intact protein mass. The university MS facility has two MS systems capable of intact protein mass characterization, an old Waters Q- Tof MS system soon to be decommissioned and a high res-Q-TOF Lumos system. This facility unfortunately is not able to provide the daily or weekly access for analysis of intact protein mass needed by the center and they cannot accommodate walk up service from center staff. Here, we request funds to acquire a benchtop MS system, 6230B TOF MS system with ESI source and 1290 LCMS system with autosampler from Agilent. A center housed MS system capable of intact protein mass analysis would allow us to quickly evaluate proteins from the center and DBP labs. We expect the instrument will serve 50-100 researchers per year, including all personnel in the center, DBP collaborators and 2 workshops of 20 people per year. A permanent center staff member will be assigned to MS equipment maintenance and user training. The in-house ability to run and process our own intact protein MS analysis on a daily basis will greatly accelerate the center mission regarding GCE technology development, optimization and dissemination for DBPs and GCE trainees. The summary of our original grant is included in our research strategy document.

装备补充摘要 俄勒冈州立大学 (OSU) 的 GCE4All 生物医学技术开发和传播中心 致力于优化、开发和广泛传播遗传密码扩展 (GCE) 技术 – 细胞翻译工程以表达含有非规范氨基酸 (ncAA) 的蛋白质。 预计寿命≤15年，该中心的使命是有效扩展现有的GCE技术，以方便 供非专业人士使用，并通过广泛的教育、有效的培训和通过 通过已建立的存储库提供对优化技术的可持续访问——实现强大的 GCE 随着我们构建新的 GCE 编码，这些方法将成为生物医学研究人员广泛使用的标准工具。 系统，量化 ncAA 蛋白的完整质量对于验证 ncAA 的位点特异性编码至关重要， 验证天然氨基酸的错误编码不会造成保真度损失，并评估双正交 然而，该中心没有表征完整蛋白质质量的设备。 该大学的 MS 设施拥有两个能够进行完整蛋白质质量表征的 MS 系统，即一台旧的 Waters Q- Tof MS 系统即将退役，而高分辨率 Q-TOF Lumos 系统不幸的是。 无法提供中心所需的每日或每周的完整蛋白质质量分析，并且他们 无法提供中心工作人员的上门服务 在此，我们请求资金购买台式 MS 系统， 配备安捷伦 A 中心配备 ESI 源的 6230B TOF MS 系统和配备自动进样器的 1290 LCMS 系统。 能够进行完整蛋白质质量分析的 MS 系统将使我们能够快速评估中心蛋白质 我们预计该仪器每年将为 50-100 名研究人员提供服务，包括该实验室的所有人员。 中心、DBP 合作者和每年 20 人的 2 个研讨会将有一名常驻中心工作人员。 分配给MS设备维护和用户培训我们自己完整的内部运行和处理能力。 每天进行蛋白质 MS 分析将大大加快有关 GCE 技术的中心任务 为 DBP 和 GCE 学员开发、优化和传播。 我们原始资助的摘要包含在我们的研究策略文件中。

项目成果

Quantitative Analysis and Optimization of Site-Specific Protein Bioconjugation in Mammalian Cells.

哺乳动物细胞中位点特异性蛋白质生物缀合的定量分析和优化。

• DOI: 10.1021/acs.bioconjchem.2c00451
• 发表时间: 2022-12-02
• 期刊: Bioconjugate chemistry
• 影响因子: 4.7
• 作者: Amy Ryan;Olivia Shade;Anirban Bardhan;Aleks;er Bartnik;er;A. Deiters
• 通讯作者: A. Deiters

PermaPhos Ser : autonomous synthesis of functional, permanently phosphorylated proteins.

PermaPhos Ser ：自主合成功能性、永久磷酸化蛋白质。

• 发表时间: 2021-12-14
• 作者: Zhu, Phillip;Franklin, Rachel;Vogel, Amber;Stanisheuski, Stanislau;Reardon, Patrick;Sluchanko, Nikolai N;Beckman, Joseph S;Karplus, P Andrew;Mehl, Ryan A;Cooley, Richard B
• 通讯作者: Cooley, Richard B

Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli.

将磷酸丝氨酸定点掺入大肠杆菌的重组蛋白中。

• 发表时间: 2022-11-05
• 影响因子: 0.8
• 作者: Zhu, Phillip;Mehl, Ryan A;Cooley, Richard B
• 通讯作者: Cooley, Richard B

Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions.

使用优化的表达条件获取含有基因编码磷酸丝氨酸的同位素标记蛋白质，用于 NMR。

• 发表时间: 2022-12
• 作者: Vesely, Cat Hoang;Reardon, Patrick N;Yu, Zhen;Barbar, Elisar;Mehl, Ryan A;Cooley, Richard B
• 通讯作者: Cooley, Richard B

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ.

用于定义单体 14-3-3ζ 的相互作用组的功能性、永久磷酸化蛋白质的自主合成。

• 发表时间: 2023-04-26
• 影响因子: 18.2
• 作者: Zhu, Phillip;Stanisheuski, Stanislau;Franklin, Rachel;Vogel, Amber;Vesely, Cat Hoang;Reardon, Patrick;Sluchanko, Nikolai N;Beckman, Joseph S;Karplus, P Andrew;Mehl, Ryan A;Cooley, Richard B
• 通讯作者: Cooley, Richard B