Rapid, continuous purification of proteins in a microfluidic device using genetically-engineered partition tags
Rapid, continuous purification of proteins in a microfluidic device using genetically-engineered partition tags
复制标题
DOI：
10.1039/b716462a
复制
发表时间：
2008-01-01
期刊：
影响因子：
6.1
通讯作者：
Singh, Anup K.
中科院分区：
文献类型：
--
作者：
Meagher, Robert J.;Light, Yooli K.;Singh, Anup K.
天然蛋白和重组蛋白的高通量筛选分析在生命科学研究中越来越重要，包括药物筛选和酶工程等领域。这些化验通常高度平行，每次化验需要极少量的纯化蛋白。为了满足这一需求，我们开发了一种快速、自动化的微型程序，用于从亚微升体积的大肠杆菌细胞裂解物中分离特定的蛋白质。重组蛋白质被基因标记，以驱动分配到流动的双水相系统的富PEG相，在简单的微流控设备上，该系统可以去除类似85%的污染蛋白质，以及不需要的核酸和细胞碎片。基因标签的加入大约是自动荧光蛋白AcGFP 1回收率的三倍，还显著提高了谷胱甘肽S转移酶(GST)的回收率，而野生型酶的回收率几乎为零，使用遗传标签的回收率高达40%。提取过程持续进行，从细胞裂解液到纯化蛋白只需一步，不需要昂贵的亲和试剂或麻烦的层析步骤。两相体系温和，不会破坏蛋白质的功能，从我们的微流控过程中恢复了活性酶和功能性荧光蛋白就是明证。微流控双水相萃取形成了集成的芯片实验室设备的核心组件，该设备包括细胞培养、裂解、纯化和在单个设备上的分析。
High-throughput screening assays of native and recombinant proteins are increasingly crucial in life science research, including fields such as drug screening and enzyme engineering. These assays are typically highly parallel, and require minute amounts of purified protein per assay. To address this need, we have developed a rapid, automated microscale process for isolating specific proteins from sub-microlitre volumes of E. Coli cell lysate. Recombinant proteins are genetically tagged to drive partitioning into the PEG-rich phase of a flowing aqueous two-phase system, which removes similar to 85% of contaminating proteins, as well as unwanted nucleic acids and cell debris, on a simple microfluidic device. Inclusion of the genetic tag roughly triples recovery of the autofluorescent protein AcGFP1, and also significantly improves recovery of the enzyme glutathione S-transferase (GST), from nearly zero recovery for the wild-type enzyme, up to 40% with genetic tagging. The extraction process operates continuously, with only a single step from cell lysate to purified protein, and does not require expensive affinity reagents or troublesome chromatographic steps. The two-phase system is mild and does not disrupt protein function, as evidenced by recovery of active enzymes and functional fluorescent protein from our microfluidic process. The microfluidic aqueous two-phase extraction forms the core component of an integrated lab-on-a-chip device comprising cell culture, lysis, purification and analysis on a single device.