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expression with M15 - Any special tips?? (Feb/07/2007 )

Hi, as I've been trying to express a protein in BL21 plys with pET22 and had no induction, I decided to change the system to one that members of my lab have used before.
It involves the used of M15 pREP4 strain and the pQE31 plasmid with the construction of interest. It's rather curious what happend with are protocol: it only worked the first time we used it!! wacko.gif
We tried it again and again and we didn't get expression of the protein!! that's why I changed to BL21, I was tired of
trying the same protocol and getting no results....But as the BL21 system didn't work either I decided to give it a last tried with the first one.

The protocol I have involves the following steps:

1- transform M15 with pQE31-my protein an pour on an LB agar dish ON
2- On the next day take some colonies and grow ON in 3ml LB each
3- To check expression:
- Grow in 3ml LB a 1/10 dilution of each culture for 2 hs
- Then induced a 1,5ml alicuote with IPTG 10mM final [ ] and leave 1,5ml without induction as control
- Grow at 37º for 3:30hs
- Centrifugue both samples and resuspend the pellet in 40ul Laemmli
- Hit the samples at 90º -100º for 5 minutes
- Run in SDS PAGE
- Check if there is overexpression
...And if there is expression, I grow a larger culture ON (50 ml) to do the induction of more volumen on the following day.
I add the antibiotic to the medium in every growing step and we used 10mM IPTG because someone in my lab once checked the best concentration...the result was that between 1 and 10mM IPTG you have good induction. (in fact in one of the many times I did the same expression protocol I used 1mM and of course, as I said before, I had no expression of the protein.........so maybe could that be the reason for the negative result??)

I don't know what might be happening; if the protein is toxic or there is a problem with the construction it wouldn't have expressed the first time........so I don't know!! glare.gif

If anyone can give me any opinion! wink.gif

-biotech!-

I can only share my own experience cause I am dealing with pQE series as well.
I have tried M15 and the other one SG1?????. Both with pREP4 strain. None works. But I changed to BL21, it seems to have some expressed protein. And my induction period is longer than yours, around 5 hours.

Maybe you can try at lower temperature.
Previously you were mentioned that your other lab member used BL21 Plyss. I think that competent cells are meant for toxic protein. Hope it helps...

-timjim-

QUOTE (timjim @ Feb 8 2007, 12:03 PM)
I can only share my own experience cause I am dealing with pQE series as well.
I have tried M15 and the other one SG1?????. Both with pREP4 strain. None works. But I changed to BL21, it seems to have some expressed protein. And my induction period is longer than yours, around 5 hours.

Maybe you can try at lower temperature.
Previously you were mentioned that your other lab member used BL21 Plyss. I think that competent cells are meant for toxic protein. Hope it helps...



Another question: before doing the competent cells, how do you start the culture?...I mean for preparing the competents, do you do an ON liquid culture of M15 (or BL21) directly from frozen bacteria (-70º) or you do an agar plate from the frozen ones and then pick a colony to do the liquid ON, to prepair the competent bacteria on the following day??
So far I always take some of the frozen bacteria and directly put the tip in LB media to grow on, could that be the reason for not having good expression results?? I don't think so, but who knows!

-biotech!-

I usually grow them on agar plate from glycerol stock (-70). And only pick 1 colony to do culture ON. It took me few days to do it actually.

I don't really think that could be the reason cause if you were able to transform inside, it doesnt really matter. The competent cells have your desired plasmid. So what's gonna do with your expression. I might be wrong though. wink.gif

-timjim-