I am trying to express recombinant proteins using Bac To Bac expression system (invitrogene). However, I am encountering two problems I wasn't able to solve yet.
1) I am not able to express satisfactory amount of two of my proteins over 120kDa. I've tried to change cell line and viral stock titre but I am still not able to detect my proteins by Coomasie staining and even with Western blot the protein is hardly visible.
2) even though I am expressing intracellular proteins and also included low amount of FBS into the expression experiments, most of my proteins are getting degraded (some of them just slightly, some of them a lot). Can anyone explain me why and what to do about it? Thanks a lot for all suggestions.
expression levels could vary a lot from protein to protein
- how do you purify them?
I would try double tag (N-term Strep tag, C-term His tag), this way only whole
length protein will be purified.
I need some details ..before answering your question...
What is your buffer composition? pH etc
What is the expected PI of our protein?
How you checked expression of your protein ( It might going to the pellet/ inclusion bodies)
What was the inducer?
These information will give clue about nature/ stability and level of expression of protein.
Hola Vera I suposse that at least you use a MOI bigger than 3 ( 10 the best) and let the cells express untill they dead (72-96h) or you see all infected under microscope. I have expressed lots of proteins in this system and only saw some degradation when the amount of protein of interest was very high. Good luck
I didn't try to purify yet since I was never able to get enough protein to do so. I express the protein tagged by C term His in Hi5 or Sf9 cell line (tried both of them, didn't seams to be much different) using Bac To Bac expression system from invitrogen. I just lysed the cells and analyzed the protein expression by SDS page and subsequent Coomasie staining, and also the whole cell lysate by Western blot with Anti His C term antibody (invitrogen). I suppose it should be specific enough to distinguish His C term tag clearly? Anyway, the biggest problem for me is now that the protein doesn't seams to express very well.
The PI of the protein is over 9.5. For infection do you thing higher MOI would help? Aren't there also higher chance that the protein would go wrong? I am a bit confused about the MOI to be honest. Some researchers recommend high MOI, some of them rather lower one... Based on some papers I've read before I've started the experiments I've chosen MOI 3 since they have said that this would be sufficient enough to express a lot of high quality protein, decrease the risk of expressing proteins containing mutations and also prevent premature cleavage of the protein.
Thanks a lot for all the responses and suggestions.
I would try to purify anyway. In crude lysates you need pretty high level of
expression to see your protein.
Dear Vera,
there may be multiple reasons for the low expression. Regarding the proteolysis issue, there are new bacmid versions with deletions of two baculoviral genes one of which is a protease responsible for some of the observed degradation. You can get more information on this here: http://www.embl.fr/multibac/multiexpression_technologies/multibac/index.html
Lucky for you, Czech Republic is now a member country of EMBL and if you want we could try to help you with your baculovirus expression in our facility. Just send me a message via our web page:
http://www.embl.de/pepcore/pepcore_services/contact/index.html
I am not a fan of the Bac-to-Bac system, to easy to generate non-recombinant virus and then amplify the non-recombinant virus resulting in low protein expression. I use either BaculoGold DNA (no non-recombinant Bacs in my hands) or flashBac DNA (no non-recombinant Bacs in anyone's hands due to the design), see attached paper.
Thank you for all of your responses. I will try to prepare new viral stocks and increase MOI to 10 and I will include some additional steps. Hope I will manage somehow. To be honest I don't think Bac To Bac is a problem. I've tried to express this protein in bacterias as well lately (cloned into pBSK3 expression vector) and it is also a living hell. Anyway, thanks again for all the help.
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