Sugrue R J, Cui T, Xu Q, Fu J, Chan Y C
Dengue Virus Group, Institute of Molecular and Cell Biology, National University of Singapore, Singapore.
J Virol Methods. 1997 Dec;69(1-2):159-69. doi: 10.1016/s0166-0934(97)00151-1.
The dengue virus envelope protein was expressed as a GST fusion protein using E. coli and P. pastoris as expression hosts. In E. coli the recombinant E protein is expressed initially as a soluble 81 kDa GST fusion protein. Treatment of the fusion protein with thrombin released a 55 kDa protein, which is the expected size for correctly processed, non-glycosylated recombinant E protein. The antiserum from animals immunised with this recombinant E protein was found to specifically recognise the dengue virus E protein in virus-infected cells, thus demonstrating the immunogenic nature of the recombinant E protein. This expression system allowed production of up to 2 mg of purified recombinant E protein from a 1 1 bacterial culture. In contrast, expression of this GST fusion protein in P. pastoris is associated with extensive proteolytic degradation of the recombinant E protein. However, this proteolytic degradation was not observed in the truncated E protein sequences which were expressed. One of these recombinant fusion proteins, GST E401 was secreted into the culture medium at levels of up to 100 microg/l of growth medium.
利用大肠杆菌和巴斯德毕赤酵母作为表达宿主，将登革病毒包膜蛋白表达为谷胱甘肽S-转移酶（GST）融合蛋白。在大肠杆菌中，重组E蛋白最初表达为可溶性的81 kDa GST融合蛋白。用凝血酶处理该融合蛋白可释放出一个55 kDa的蛋白，这是正确加工的非糖基化重组E蛋白的预期大小。用这种重组E蛋白免疫动物得到的抗血清能特异性识别病毒感染细胞中的登革病毒E蛋白，从而证明了重组E蛋白的免疫原性。该表达系统从1升细菌培养物中可产生高达2毫克的纯化重组E蛋白。相比之下，在巴斯德毕赤酵母中表达这种GST融合蛋白时，重组E蛋白会发生广泛的蛋白水解降解。然而，在所表达的截短E蛋白序列中未观察到这种蛋白水解降解。其中一种重组融合蛋白GST E401分泌到培养基中的水平高达每升生长培养基100微克。
