Mammalian protein expresion in bacteria - Which plasmid and strain to use (May/22/2007 )
I'm just starting with my protein extraction work. At present we are using pMMB66 plasmid to express a fluorescence protein in bacteria JM109 and it works fine (probably there are methods to improve the final amount obtained).
My plan is to insert a human protein with GFP in pMMB66 and use JM109 to extract the protein. Should I have any problem with this? What things should I take into consideration? It is a small protein.
Anyone knows a good website/paper to have a look to try to improve my protein induction/extraction methodology from bacteria?
Anyone can give me a hand, pleeeeease?
What is the desired protein size?
GFP is fluorescence signal protein. What kinda of steps after running the gel? Western?
My apology, I am just trying to understand the whole situation.
Thanks for the reply. It is a variant of the GFP, so the size is 600bp.
To calculate the effectiviness of our protein extraction protocol we just measure the fluorescent signal which is approximately proporcional to the amount of the GFP-like protein present in the final solution.
Have you transform the plasmid in? Have you try to express the GFP protein?
Yes, that's exactly what I want and I would like to do in bacteria to get good amounts of the protein and be able to study it better (pH changes, for ex)
Oh ok... I am really familiar with GFP but only heard of it. So my suggestion might be really vague.
Anyway, my humble opinion will be transform it into some expression vector like BL21 though DH5A should works if the plasmid itself has strong promoter sequence. JM109 as you mentioned earlier on is ok too.
After transformation, then you have to think about how to lyse the cells to get desired protein. Detergent lysis? Or free thaw? up to you really. My bet will be sonicator. Much more efficient.
we tested many bacterias in our lab and Rosetta strain was fine for both 9kD and 97kD proteins.
Purification on HPLC was very clean