Options for protein expression? - (Oct/05/2006 )
Could anyone give me a guide as to how to express a protein I plan on purchasing as a cDNA clone from Origene? I am imagining that I will need to clone it into an expression vector for either e.coli, insect or mammalian cells? Which way is easiest/fastest/best?
it depends a little on what your downstream applications are. many proteins are expressed in ecoli fairly easily, but some proteins require quite a bit of tweaking and effort even in e coli
if I were you, I would start with reading some of the guidelines on Qiagen's website - here is a link - and see if you can get an idea of what sort of tag you want, and what to consider in terms of purification protocols before you decide what vector to use. even if you don't use Qiagen's stuff, the guides are pretty good info; just look for corresponding tags and vector systems from the vendor of your choice....this is IF you want to purify a bunch of recombinant protein?
Thanks for the advice. Yeah, that's the thing is that I don't need a lot of protein. Just enough for WB confirmation that our antibodies work. I have been using whole cell lysates up until now, which give me headaches and lots of non-specific bands. So, I was just toying with what it would take to make myself some of the proteins I am interested in. I am most familiar with transfecting mammalian cells, IP-ing, and then doing western blot, but I didn't know if there was a better or easier alternative way to get the protein made and purified. I will look at the site, thanks!
another possibilty would be an in-vitro translation kit? I haven't tried this, but I bet someone else here has a recommendation for you and it may be easiest/best for you? if you are much more worried about a clean, pure prep than you are about quantity, then an expression system with a histag vector or whatever may not be the best for your protein