Wang Y, Mukhopadhyay A, Howitz V R, Binns A N, Lynn D G
Department of Chemistry, The University of Chicago, IL 60637, USA.
Gene. 2000 Jan 25;242(1-2):105-14. doi: 10.1016/s0378-1119(99)00541-7.
A versatile expression vector utilizing a promoter of coliphage T5, P(N25) (Gentz and Bujard, 1985. J. Bacteriol. 164, 70-77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupré et al., 1997. J. Bacteriol. 179, 78-89) has been developed. This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (delta1-284, G665D) in Escherichia coli as well as in A. tumefaciens. The signal transduction proteins VirA (delta1-284, G665D) and VirG are fully functional when expressed in Agrobacterium, and the P(N25) driven expression overrides the complex transcriptional regulation present with the native promoters. This expression system will enable a more detailed analysis of the activation events in signal transduction in A. tumefaciens, and we expect it to be useful in other prokaryotes.
利用大肠杆菌噬菌体T5启动子P(N25)（根茨和布亚德，1985年。《细菌学杂志》164卷，70 - 77页）和IncW广宿主范围质粒pJB20的衍生物（博普雷等人，1997年。《细菌学杂志》179卷，78 - 89页）构建了一种多功能表达载体。该载体成功地在大肠杆菌以及根癌土壤杆菌中表达了由virG编码的根癌土壤杆菌的毒力蛋白和virA的一个突变等位基因virA（delta1 - 284，G665D）。信号转导蛋白VirA（delta1 - 半胱氨酸，G665D）和VirG在根癌土壤杆菌中表达时功能完全正常，并且P(N25)驱动的表达超越了天然启动子存在的复杂转录调控。这个表达系统将能够更详细地分析根癌土壤杆菌信号转导中的激活事件，并且我们预计它在其他原核生物中也会有用。
