Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
J Biomol NMR. 2012 May;53(1):43-51. doi: 10.1007/s10858-012-9619-4. Epub 2012 Mar 15.
Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [(15)N,(1)H]-HSQC NMR.
大量生产人类蛋白质是结构生物学中常见的瓶颈。在这里，我们描述了一种基于大肠杆菌的无细胞系统，该系统可在与 GB1 结构域融合的 N 端构建体中产生毫克级别的人类蛋白质，其翻译效率显著提高。我们使用了一种新生成的大肠杆菌 BL21（DE3）RIPL-Star 菌株，该菌株含有一种活性降低的变体 RNase E 和过量的稀有密码子 tRNA，并且缺乏 lon 和 ompT 蛋白酶活性。在表达系统的实施中，我们使用了新制备的细胞提取物。与连续交换表达相比，使用此设置的分批无细胞表达更经济，反应混合物每毫升的产量为 0.2-0.9 毫克纯化蛋白。通过 2D [（15）N，（1）H] -HSQC NMR 证明了所获得的蛋白质的天然折叠。
