Democratizing the rapid screening of protein expression for materials development
Democratizing the rapid screening of protein expression for materials development
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DOI：
10.1039/d2me00150k
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发表时间：
2022-10-28
影响因子：
3.6
通讯作者：
Olsen，Bradley D.
中科院分区：
文献类型：
--
作者：
Morris，Melody A.;Bataglioli，Rogerio A.;Olsen，Bradley D.
蛋白质材料的功能、结构和机械性能使其非常适合生物传感器和生物材料等一系列应用。与传统的聚合物合成不同，它们的序列是被定义的，对于重组蛋白来说，是由所选的 DNA 序列决定的。随着DNA合成在过去二十年的快速发展，蛋白质材料开发的限制瓶颈是蛋白质表达的经验优化。在此，开发了一种低成本、自动化、高通量的组合蛋白表达平台，用于在 96 孔板格式中测试 DNA 载体和大肠杆菌 (E. coli) 菌株的排列。使用 600 nm (OD600) 的光密度监测生长和表达以测量生长，使用 Bradford 测定法确定总蛋白浓度，并使用斑点印迹测定法测定目的蛋白的浓度。着眼于没有成套生物合成设备的研究人员的可及性，基于自动相机的检测通过比浊法验证了 OD600 检测，并通过比色法验证了 Bradford 检测。高产表达条件一周内即可确定。值得注意的是，在一些情况下，以前无法表达的蛋白质能够以可行的产量成功表达。总的来说，建立了一种克服蛋白质材料开发中长期存在的合成挑战的有效方法，这将加速材料创新。
The function, structure, and mechanical properties of protein materials make them well-suited for a range of applications such as biosensors and biomaterials. Unlike in traditional polymer synthesis, their sequences are defined and, in the case of recombinant proteins, dictated by the chosen DNA sequence. As DNA synthesis has rapidly progressed over the past twenty years, the limiting bottleneck in protein materials development is the empirical optimization of protein expression. Herein, a low-cost, automated, high-throughput, combinatorial protein expression platform is developed to test permutations of DNA vectors and Escherichia coli (E. coli) strains in a 96-well plate format. Growth and expression are monitored with optical density at 600 nm (OD600) to measure growth, Bradford assays to establish the total protein concentration, and dot blot assays to determine the concentration of the protein of interest. With an eye toward accessibility for researchers without suites of biosynthetic equipment, automated camera-based assays are validated for the OD600 assay, via turbidimetry, and the Bradford assay, via colorimetry. High-yield expression conditions can be determined within a week. Notably, in several cases, previously un-expressible proteins are expressed successfully in viable yields. Collectively, an efficient approach to overcoming long-running synthesis challenges in protein materials development is established, which will expedite materials innovation.