Hi…researchers…..
I have cloned a mature serine protease into pET30a vector and tried to express in E.coli, BL 21 (DE3). I have followed the expression setup as given below:
Day 1: grown overnight culture in LB broth at 28 degrees
Day 2: 1% inoculums was prepared for starter at 27 degrees, after 2 Hrs, the OD@600nm was reached to 0.5-0.6. The culture was then induced with 0.2-0.6 mM IPTG at 25 degrees. The growth of un-induced culture was 1.2 OD and induced was 0.8 OD after 3 Hrs. However, no difference between Un-induced and induced was observed. What I should do further?
My protease is toxic to E.coli? or what?
Kindly guide me............advance thanksssss
Hi Mani,
From the information you have given I think it is highly likely you have a 'toxic' protein. Expressing an active protease is always going to be difficult. The poor growth of your un-induced culture would seem to indicate that your protein is having a serious affect on the cells - from a very low level of leakiness.
What to do?
Grow the cells to an OD of 2 to 3 (if possible) before induction. Test harvest points every hour to see if you have any expression. Do not expect high levels of expression but you might get something.
Perhaps try adding some protease inhibitors to the cultures (I've never done this so don't know if it will work).
or
Expression of the immature protein followed by maturation after purification might be one route.
-Richard
Respected Professor Richard Christison,
Greetings!!!!
Thanks for your message. I have already expressed immature protease (37 kDa Serine protease from Bombyx mori, (Accession number: cDNA -an-0779; AB434653), however, It was not get activated, Is there any tools for activate them? Yes, a report was there, it can activate by mixture midgut lysate. But I don’t know what is really playing for activation (Kaji et al., 2009).
Is it right procedure to induce 2 or 3 OD cells for expression while compared with 0.4 OD of cells?
Any how I will try and post the comments at the earliest.
Thanks...........
Hi Mani,
The idea behind high OD induction is that you have increased the biomass, so that on induction you get a small amount of protein from many cells. Thus you get a little from many cells, not a little from few cells. It is used mainly when you cannot get continuous expression over time, due to the protein affecting the cells. You are unlikely to ever get a high level this way but could obtain a useable amount. Scaling up the volume of the culture may also be required.
I'd advise you to find out all you can on your and related protease as to activation. Many require cleavage of the protein, it may be possible to insert a known protease site to enable activation, depending on the protein.
-Richard
p.s. By the way, it's not a good idea to give people titles they don't possess.
Respected Professor!!!!Greetings!!! Thanks for your comments, I am doing the experiments as per your suggestion and I will post the comment the at the earliest.Thanking You,Yours Sincerely,M.Kannan
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