کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2021905 | 1069267 | 2007 | 9 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Differential expression of the Ebola virus GP1, 2 protein and its fragments in E. coli Differential expression of the Ebola virus GP1, 2 protein and its fragments in E. coli](/preview/png/2021905.png)
Bacterial expression platforms are frequently used for the expression and production of different recombinant proteins. The full length Ebola virus (EBOV) GP1, 2 gene and subfragments of the GP1 gene were cloned in a bacterial expression vector as a C-terminal His6 fusion protein. Surprisingly, the full length EBOV GP1, 2 gene could not be expressed in Escherichia coli. The subfragments of GP1 were only expressed in small amounts with the exception of one small fragment (subfragment D) which was expressed at very high levels as inclusion bodies. This was seen even in the in vitro translation system with no expression of full length GP1, 2, GP1 subfragments A and C and low level expression of subfragment B. Only the subfragment D showed high level of expression. In E. coli (Top10), the recombinant GP1 subfragment D protein was expressed exclusively as an insoluble ∼25 kDa His6 fusion protein, which is the expected size for a non-glycosylated recombinant protein. The IMAC purified and refolded non-glycosylated protein was used to immunize mice for the development of monoclonal anti-EBOV antibodies which successfully yielded several monoclonal antibodies with different specificities. The monoclonal and polyclonal antiserum derived from the animals immunized with this recombinant GP1 subfragment D protein was found to specifically recognize the full length glycosylated EBOV GP1, 2 protein expressed in mammalian 293T cells, thus, demonstrating the immunogenicity of the recombinant subfragment.
Journal: Protein Expression and Purification - Volume 54, Issue 1, 1 July 2007, Pages 117–125