Posted 13 May 2009 - 10:43 PM
I'd like to express and purify a heterodimeric kinase in bacteria. Is it possible to put both genes into one vector, the second gene with a ribosome binding site in front? The first subunit has then a 6xHis tag, can I purify the whole kinase with this? Would you think that this kinase might work in bacteria, ie. when I further transfect a kinase target, would that be phosphorylated in the bacteria?
Posted 15 May 2009 - 10:44 PM
Posted 16 May 2009 - 07:30 PM
Unfortunately, our lab budget is a little tight, so I was hoping to circumvent buying a new vector (for example the pETDuet from Novagen) by creating it myself... There is some literature about this, e.g. PMID 19017543, where they expressed two proteins just by integrating a second ribosome entry site. Anyone some experience?
I guess the other questions are not easy to answer, I might need to try and find out myself...
Thanks again and good luck to all of you.