Development of a high throughput platform for screening directed evolution libraries

在实验室中蛋白质的受控进化是一种有价值的生物医学工具，用于获得生物分子， 工业、治疗和研究应用。这个过程，也被称为定向进化，允许一个人 利用大自然赋予其特权生物分子的特异性和选择性来构建非天然的 否则化学合成产生的效率低或费力的产物。虽然这个过程是 令人难以置信的强大，现有的瓶颈是随后筛选这些高水平的变异体。 价值产品。定向进化过程通常产生数百至数千个突变体或文库， 进行生化分析。在一些情况下，可以使用荧光报告系统或生物活性测定。 然而，作为一般的生物化学读数，这并不能说明具体的化学转化 针对不同的小分子目标。当高价值的化学产品是这些指示的主题时， 在进化实验中，研究人员采用多种正交分析技术，包括： 高效液相色谱法（HPLC）;气相色谱法（GC）;质谱法（MS）;和核 磁共振（NMR）。当成千上万的变体需要 即使将变异体合并到精心挑选的组中，也需要相当大的努力进行色谱分析。 考核此外，这些方法中的许多方法可能不够敏感或具体， 检测所需产物的低滴度生产。基于这些缺点的筛选 平台，我们建议利用我们实验室的现有优势，开发高通量， 用于筛选生物活性的定向进化文库的特异性和灵敏的质谱分析平台 化工产品不需要色谱分离。麦金尼实验室擅长合成 化学和生物化学，并专门研究α-酮戊二酸依赖性双加氧酶 以克级构建神经活性红藻氨酸。桑切斯实验室在天然产物方面 发现和质谱技术，如成像质谱和串联质谱 光谱法这些各自的优势将使我们能够开发一个创新的管道， 定向进化文库成员优先考虑将化学物质导向类海人藻毒素环的变体 谷氨酸受体激动剂和拮抗剂。我们的管道将允许前所未有的测量， 化学特异性，并广泛适用于任何寻求进行定向进化的群体。 ●目前的定向进化筛选平台耗时或通量低 ●我们团队的综合专业知识是高度跨学科的 ●质谱和捕获离子迁移谱允许高维度 直接从突变菌落中进行测量，而不依赖色谱技术

The controlled evolution of proteins in the laboratory is a valuable biomedical tool for accessing biomolecules for industrial, therapeutic and research applications. This process, also known as directed evolution, allows one to employ the specificity and selectivity that Nature imbues within its privileged biomolecules to construct unnatural products that would otherwise be inefficient or laborious to generate chemosynthetically. While this process is incredibly powerful, an existing bottleneck is the subsequent screening of the resulting variants for these high value products. The directed evolution process typically generates hundreds to thousands of mutants or library members for biochemical analysis. In some cases, fluorescent reporter systems or bioactivity assays can be employed as a general biochemical readout, however, this does not inform on specific chemical transformations towards diverse small molecule targets. When high value chemical products are the subject of these directed evolution experiments, researchers employ multiple orthogonal analytical techniques, including: high performance liquid chromatography (HPLC); gas chromatography (GC); mass spectrometry (MS); and nuclear magnetic resonance (NMR). This becomes time and infrastructure intensive when thousands of variants need to be evaluated; even if variants are pooled in curated groups, considerable effort is needed for chromatographic assessment. Additionally, many of these methodologies may not be sensitive or specific enough to necessitate detection of low titer production of the desired product(s). Based on these shortcomings of the screening platforms, we are proposing to leverage our labs’ existing strengths to develop a high-throughput, specific, and sensitive mass spectrometry platform to screen directed evolution libraries for bioactive chemical products without chromatographic separation. The McKinnie lab has expertise in synthetic chemistry and biochemistry and has specifically worked on the α-ketoglutarate-dependent dioxygenase enzyme to construct neuroactive kainic acid on the gram scale. The Sanchez lab has expertise in natural product discovery and mass spectrometry techniques such as imaging mass spectrometry and tandem mass spectrometry. These respective strengths will allow us to develop an innovative pipeline for screening thousands of directed evolution library members to prioritize variants that direct the chemistry towards kainoid-ring glutamate receptor agonists and antagonists. Our pipeline will allow for unprecedented measurements in chemical specificity and be broadly applicable for any groups looking to conduct directed evolution. ● Current directed evolution screening platforms are time-consuming or low throughput ● The combined expertise of our team is highly interdisciplinary ● Mass spectrometry and trapped ion mobility spectrometry allow for high dimensionality measurements directly from mutant colonies without reliance on chromatography techniques