Murthy T V S, Wu Weilin, Qiu Q Q, Shi Zhenwei, LaBaer Joshua, Brizuela Leonardo
Harvard Institute of Proteomics, 320 Charles street, Cambridge, MA 02141, USA.
Protein Expr Purif. 2004 Aug;36(2):217-25. doi: 10.1016/j.pep.2004.04.002.
Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159 kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions. Most of the expressed proteins showed yields greater than 500 ng after a single affinity purification step from 50 microl in vitro protein synthesis reactions. The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4 h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.
对63种大小在18 - 159 kDa范围内的铜绿假单胞菌蛋白质进行了无细胞细菌系统中的表达测试。63种蛋白质中有51种可以在变性条件下表达并部分纯化。在50微升体外蛋白质合成反应经过单次亲和纯化步骤后，大多数表达的蛋白质产量超过500纳克。一个人可以在4小时内完成96孔板格式的体外蛋白质表达加纯化，并通过SDS - PAGE对蛋白质进行分析。体外和体内表达的比较表明，尽管产量较低且蛋白质制备纯度较低，但细菌体外蛋白质表达结合单步亲和纯化可为蛋白质表达和溶解性克隆的高通量筛选提供一种快速、高效的替代方法。
