Thank you for your kind advices. In this case, pGEX4T3 vector also expressed inserts constitutively. http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm
I also thought that target protein may have tocicity on host strain. But, why pGEX4T3-target protein was not show the same phenomena. Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and BL21(DE3)pLysS. Is this try may helpful to solve the this kind of problems? Thanks for your valuable comments. Genie 2011/7/14 aaleshin <aales...@burnham.org> > I never used pET20b, but I found on the Internet that it expresses inserts > constitutively. Since the cloned protease should be toxic to cells, its > constitutive expression might prevent the formation of colonies. But there > might be millions of other reasons. Why did you use pET20b? > > http://www.biovisualtech.com/bvplasmid/pET-20b%28+%29.htm > > Alex > > > > > > On Jul 13, 2011, at 5:54 PM, Wonjin Bae wrote: > > Hi, all > > Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and > pGEX4T3 vector. > Then, these two construct were transformed to BL21(DE3) expression host. > DNA sequencing results were accurate. > > In case of pGEX, many colony was formed and shown the high level of > expression. > But, colony was not shown in pET20b case. (no one colony) > In case of mock-pET20a vector transformed very well. > > What's the problems? I never experieced transfomation problems. > Does anyone know how to solve the this problems? > > Thanks a lot !! > Genie, > > > -- Jin Soo Bae 790-784 room 204, Dept of Life Science, POSTECH, san31, Hyoja-dong, Nam-gu, Pohang, Gyungbuk, Korea tel:82-54-279-8627 fax:82-54-279-8111
