Golz S, Birkenbihl R P, Kemper B
Institut für Genetik, Universität zu Köln, Germany.
DNA Res. 1995 Dec 31;2(6):277-84. doi: 10.1093/dnares/2.6.277.
Using PCR, we cloned T4 gene 49, which encodes the endonuclease VII, and the inactive mutant gene 49 amE727 into vector pET-11a. In combination with Escherichia coli host strain BL21 (DE3), this system provided excellent repression of the expression of the highly toxic protein before induction with IPTG. After induction, the proteins were made in high quantities while remaining soluble. Dilution of the crude lysate at 1:10,000 continued to show a highly specific activity in the case of the wild-type enzyme. The protein was purified to homogeneity with a recovery of 33% using two chromatography steps. The yield was 20 times higher and the specific activity 500 times higher than that obtained by using the previously published protocol.
我们利用聚合酶链式反应（PCR）将编码核酸内切酶VII的T4基因49以及无活性的突变基因49 amE727克隆到载体pET-11a中。该系统与大肠杆菌宿主菌株BL21(DE3)相结合，在使用异丙基-β-D-硫代半乳糖苷（IPTG）诱导之前，能出色地抑制高毒性蛋白的表达。诱导后，蛋白质大量产生且保持可溶状态。对于野生型酶，将粗裂解物稀释10000倍仍显示出高特异性活性。通过两步色谱法将该蛋白质纯化至同质，回收率为33%。与使用先前发表的方案相比，产量提高了20倍，比活性提高了500倍。
