Daegu Gyeongbuk Institute of Science and Technology, Korea.
BMB Rep. 2010 May;43(5):319-24. doi: 10.5483/bmbrep.2010.43.5.319.
Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value.
为了鉴定任何瓶颈以及影响目标纯化的因素，对基于大肠杆菌细胞的真核蛋白纯化实验获得的实验生物信息学数据进行了分析。所有的目标都被表达为 His 标签的麦芽糖结合蛋白（MBP）融合构建体，并通过固定金属亲和层析（IMAC）进行初步纯化。随后，通过 TEV 蛋白酶切割将目标从 His 标签的 MBP 中分离出来，然后进行第二次 IMAC 分离。在总共 743 次纯化试验中，有 342 次获得了超过 3 毫克的目标蛋白用于结构研究。目标蛋白纯化失败的主要原因是 TEV 蛋白酶切割效果不佳。目标蛋白纯化的总体成功率随着半胱氨酸含量或等电点（pI）的增加呈线性下降。pI 与总体成功率之间的这种模式强烈表明，pI 应该与阈值一起纳入目标评分标准。
