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Trouble with protein expression in E.coli


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#1 res_org

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Posted 16 June 2009 - 09:04 AM

I have generated some constructs in pETTOPO vector. DNA sequence confirms the insert in -frame.

My protein is 9-11 kDa. I have tried expression in BL-21 DE3 star and BL Ai cells using IPTG and arabinose respectively. I have checked both in soluble and pellet , but I do not see any expression of my proteins.

I am using 15% gels and stainning them with coomassie.

Please help!

Thanks

#2 pras45

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Posted 16 June 2009 - 12:51 PM

Try doing some mini-optimization using couple set of ~100 ml of LB or TB where you analyze variables like
1) Temperature 30, 25 or RT FOR post IPTG induction
2) Concentration of IPTG , did you check if your construct has IPTG inducible promoter. You could go higher but optimum is ~0.5mM
3) Pre-induction OD should be about 0.6 at 600nm, try cooling your pre-induced bacterial media at 4 degree for about 15 mins before hitting it with IPTG
4) Length of post induction time from 4 hrs - 12 hrs ,
5) Take sample of pre and post induction sample and try running those on gel before lysing your pellet, this will save a lot of time.
6) Try looking for the vector manual regarding its optimal expression, for example GE's pGEX vector has info on its maximal expression.
7) Use a positive control eg a protein that the lab has been sucessfully expressed and purified.

GOOD LUCK

#3 res_org

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Posted 18 June 2009 - 09:21 AM

Try doing some mini-optimization using couple set of ~100 ml of LB or TB where you analyze variables like
1) Temperature 30, 25 or RT FOR post IPTG induction
2) Concentration of IPTG , did you check if your construct has IPTG inducible promoter. You could go higher but optimum is ~0.5mM
3) Pre-induction OD should be about 0.6 at 600nm, try cooling your pre-induced bacterial media at 4 degree for about 15 mins before hitting it with IPTG
4) Length of post induction time from 4 hrs - 12 hrs ,
5) Take sample of pre and post induction sample and try running those on gel before lysing your pellet, this will save a lot of time.
6) Try looking for the vector manual regarding its optimal expression, for example GE's pGEX vector has info on its maximal expression.
7) Use a positive control eg a protein that the lab has been sucessfully expressed and purified.

GOOD LUCK



Thanks for your suggestions, Yes, my construct has IPTG inducible promoter. I have few questions:

how does cooling the pre-induced culture helps in expression before adding IPTG?

In your suggestion # 5, do you mean to say, just use the culture straight for SDS-PAGe analysis?

Thanks again for your help !




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