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

设备补充摘要 俄勒冈州立大学(OSU)GCE4所有生物医学技术开发和传播中心 用于优化、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。在其 预计≤的寿命为15年，该中心的使命是有效地扩展现有的通用计算环境技术，以实现 由非专业人员使用，并通过广泛的教育、有效的培训和 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 成为生物医学研究人员标准的、广泛使用的工具的方法。当我们构建新的GCE编码时 系统中，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， 以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 蛋白质上的标记反应。然而，该中心没有设备来表征完整的蛋白质质量。 大学的MS设施有两个能够完整的蛋白质质量表征的MS系统，一个老式的Waters Q- TOF MS系统即将退役，以及高Res-Q-TOF Lumos系统。不幸的是，这个设施 不能每天或每周提供中心所需的完整蛋白质质量的分析，并且他们 无法容纳中心工作人员提供的上门服务。在这里，我们请求资金来购买台式MS系统， 6230B TOF MS系统，带ESI源；1290 LC MS系统，带安捷伦自动进样器。一个有住房的中心 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的蛋白质 和DBP实验室。我们预计该仪器每年将为50-100名研究人员提供服务，包括 中心、DBP合作者和每年20人的2个研讨会。一名常设中心工作人员将 负责MS设备维护和用户培训。内部运行和处理我们自己的能力完好无损 每天进行蛋白质MS分析将大大加快GCE技术的中心任务 为DBP和GCE受训人员开发、优化和传播。 我们最初拨款的摘要包含在我们的研究战略文件中。

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.