Prokaryotic and Mammalian Expression Vectors - Why do they have to be different? (Jun/07/2005 )
I am trying to come up with an explanation why expression vectors for use in bacteria and mammalian cells need to be different? Is it something to do with the promoter? I can't quite seem to formulate it in my head. In other words, can't you use a bacterial expression vector (pGEX etc) to transfect a mammalian cell line (like COS7 or HeLa) to express your gene (for staining etc)? or vice versa?
I hope the question makes sense. Thank you.
prokaryotic and eukaryotic transcriptional machinery is different and one is not necessarily recognized by the other. All of your regulatory information will be different; pre-and post-transcriptional modification will be different as well.
one word: introns
specific structures of things like polymerase and DNA-binding proteins and other proteins involved in the transcriptional machinery are not the same from prokaryote to eukaryote and their recognition sequences will not be the same either.
if you get a basic microbiology or molecular biology text, there should be an intro chapter on prokaryotic genetics and another with eukaryotic genetics, with compare/contrast information. this should straighten it out for you.
in principle the ori site in prokaryotic vectors will not be recognised by eukaryotic hosts. and vice versa
all ur translation products from the vector would be functional only if the host is suitable due to post translational modification and regulatory mechanisms
in principle proks and euks are very different
Different expression vectors have different elements for protein expression.
A bacterial expression vector can express protein in bacteria only. It usually has pET or pBAD promoter and SD sequence.
A mammalina expression vector can express protein in mammalian only. it usually has pCMV or pEF and Kozak sequence.
Bacteria expression is easier and higher yield.
Mammalian expression is more functional and more soluble.
If you have novel protein, you might probably need to try it out.