HI,
i am trying to express a protein of 46 kda, using pRESTC vector. expressed in both BL21 DE3 and BL21 DE3 pLYS stains.
there is very little expression and i see little in uninduced also in BL21
and no difference in BL21 pLys strain
any suggestions -how to improve the expression of the protein.
induction done at 1mM IPTG and culutre grown overnight at 28 degrees
regards
aparna
Dear Stanley,
thanks for your reply.
i have earlier cloned into pET22b vector, induction was around 5-8 mg/litre, purification yield was very low, some how the protein was not binding to ni-agarose matrix. so i thought might be his at the C-terminus is not accessible, i cannot use urea or so, as this is kinase.
literature says that this protein in toxic, however i did not see reduction in the cell growth in the overnight growth.
i have used lower temperature to prevent it going into inclusion bodies.
i am trying it with N-terminus GST tag, waiting on th sequencing results.
i just have these two stains availabe and now i cannot buy any strain or any vector like bicistronic,
i am waiting to pick a good clone and then do time course and different concs of IPTG etc
do you think addition of glucose helps?
thanks
aparna
Hi
Stanley said everything , just a comment
In my experience, if the expressed protein is toxic, it induces E.coli lysis.
In your cases, Aparna, I think that your kinase is only unsoluble and it is trapped in inclusion bodies. In a point of view, inclusion bodies protects proteins from proteases digestion, I also have seen many papers describing enzyme activities (kinases too) of His-tagged proteins refolded after purification by denaturing protocols, look for them in pub-med.
I would try if the yield of the protein expressed at 37 °C is good, and you have an assay of activity easy to do.
For the expression in E.coli, it is very useful to know the hydrophobic-hydrophylic profile of the protein. For ex, it is impossible express in E.coli proteins with long hydrophobic stretch (15-20 aa), in this case it is convenient remove it. Hydrophilic regions are easily expressed in E.coli but sometime can be digested from proteases and you get very low yield.
Kinases are ancient proteins they share domains, you can do a prevision (Blast) on the domains function and chose what you need
Good luck!
Paola
what i am loading on the gel currenlty is the whole cell lysate (whole cells lysed in SDS buffer), do not know at this stage atleast if the expressed protein is in inclusion bodies
really ....not so important...
it is depent from the protein expressed
if the protein is stable or in inclusion bodiies from OD 0.6- 1 is OK
in my experience....
BL21 DE3 and BL21 DE3 pLYS stains are ment for periplasmic expression, but it depends on your protein if forms inclusion bodies. if it does, you have to extract it in denaturing conditions, else yield will be very low. also purify it under denaturing condition too. are you making whole extract or looking in periplasmic only?
did you do that in denaturing conditions?
I hope you must be having manual of BL 21, but if not you may go to:
http://www.med.unc.edu/csb/PEP/manuals/codon_plus_manual.pdf
and follow instruction given on page# 15.
with slight modification in incubation time after induction, I do it for 5-7 Hr at 37 C depends on OD
interesting ....which alcohol? Ethanol? 95° or absolute?
Could you indicate the reference?
sound so interesting, i will have a try, several of my proteins are in inclusion body, although i have dissolved with buffer+ NP-40(1%), the activity of protein is still untested.
Don't know if this question is still of any interest, but well...
For IPTG-induced protein expression you have to set up a culture with your bacteria to an OD of 0,5 - 0,7, which means the bacteria are still in log phase (and by that growing and growing) but not yet entered stationary phase.
As soon as you add IPTG the bacterial growth will drastically stop and the bacteria will start with protein production. After a couple of hours (depending on your bakteria, the expressed protein, etc. etc. ....check this by taking samples from the culture at different times after starting the IPTG-induction and analyse by SDS-PAGE) the maximum protein accumulation will be reached and this is the time when you have to harvest the bacteria. When you wait to long the degradation of the produced proteins will start and your protein output will drop dramatically.
Therefore, the best is to start a bacterial culture in the morning, let them growth till the very end of the log phase and then induce by applying IPTG for a couple of hours.
All the best,
Szymon
thanks for talking basics.. I guess that the problem was of protein going into "inclusion body".
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