Airenne K J, Kulomaa M S
Department of Biological and Environmental Science, University of Jyväskylä, Finland.
Gene. 1995 Dec 29;167(1-2):63-8. doi: 10.1016/0378-1119(95)00631-1.
A novel expression vector (pAVEX16C) has been constructed that directs the synthesis of desired polypeptides as fusions with the C terminus of chicken egg-white avidin (Avd). With this and a commercial GST gene (encoding glutathione S-transferase) fusion vector (pGEX-3X, Pharmacia), we produced Avd as fusions C- and N-terminally linked to GST in Escherichia coli. By using the Avd tail and a simple affinity purification protocol, including biotin-agarose, we were able to obtain 1-2 micrograms/ml of highly purified Avd::GST and GST::Avd from crude bacterial lysates. The produced proteins were, to a great extent, in soluble fraction when the cells were grown at 22 degrees C and disrupted with a detergent, N-laurylsarcosine. The fusion proteins could also be affinity-purified with the GST tail using glutathione-Sepharose 4B, but the yield of GST::Avd was significantly lower than when using the Avd tail. Our results therefore indicate that it is possible to produce, in E. coli, biologically active fusion proteins consisting of Avd C- or N-terminally linked with the desired protein which then can easily be purified by a simple affinity chromatography procedure.
构建了一种新型表达载体（pAVEX16C），该载体可指导合成与鸡卵清抗生物素蛋白（Avd）C末端融合的所需多肽。利用此载体和一种商业化的GST基因（编码谷胱甘肽S-转移酶）融合载体（pGEX-3X，Pharmacia公司），我们在大肠杆菌中产生了分别在C末端和N末端与GST融合的Avd。通过使用Avd尾端和包括生物素琼脂糖在内的简单亲和纯化方案，我们能够从粗制细菌裂解物中获得1-2微克/毫升的高度纯化的Avd::GST和GST::Avd。当细胞在22℃下生长并用去污剂N-月桂基肌氨酸裂解时，所产生的蛋白质在很大程度上存在于可溶部分。融合蛋白也可以使用谷胱甘肽琼脂糖4B通过GST尾端进行亲和纯化，但GST::Avd的产量明显低于使用Avd尾端时的产量。因此，我们的结果表明，在大肠杆菌中有可能产生由Avd在C末端或N末端与所需蛋白质连接的具有生物活性的融合蛋白，然后可以通过简单的亲和层析程序轻松纯化。
