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Expression problem in E.coli system using pETT22b+ from Novagen - low level of expression with unknown reason (Oct/17/2007 )

Dear friends,

I know many of you may work with pET system from Novagen to clon and express your protein in E. coli. I would be very appreciated if you can give me some sussgestions or just sharing your experience in my experimental problem.

I have clon a gen from Bacillus subtilis to pETT22b+ system purcharged from Novagen and tried to express it in E.coli BL21 and got very low expression level (LB medium is used for expression at OD620=0,7; 1mM IPTG is used for induction)
On the SDS-PAGE gel it is difficult to see the different between induced and uninduced cell, I just relised such low level of expression by comparing them to the host cell (without recombinant plasmid)
The enzyme activity showed very low activity but atleast I saw the difference between induced and uninduced experiment (induced cell is 2xhigher activity than uninduced one).
The sequence is corect in all part (the vector and the insert parts).
Is there any codon bias between E.coli and B. subtillis?
I don't know actually where is the problem.

Thanks alot in advance for all your sussgestion!



you may want to try BL21 (DE3) as the host strain


I know that E. coli and B. subtilis have virtually identical promoter consensus sequences but the translation apparatus of B. subtilis differs significantly from that of E. coli , so i agree with Amikin that you can try using BL21(DE3) or Rosetta to increase the level of expression level of protein containing rare codons.


this is an excellent review about protein expression in E.coli

hope this help too


Thank you all about the susgestion, especially to 'spanishflower' for the very useful manual of recombinant protein expression.
Last post, I have writen the wrong host expression strain, it should have been BL21(DE3) which I am using for expression.

This week I have tried to change the condition of expression (lower the temperature, lower the OD of induction time) then I got really better expression (10% of total soluble protein in E. coli) as I can see on the SDS-PAGE. Then another problem rised, the expressed protein showed very low activity although it is soluble. The protein excrete out to the medium show very good activity event it was low concentration but the one in soluble form after sonicating the cell which is susposed to be in the periplasm as the ideal did not show high activity event it was high concentration.

Do you have any idea about the inactivation of the recombinant protein at the periplasm.

I'll read the reference of Recombinant protein expression this weekend and will do the experiment to detect the distribution of recombinant enzyme next week.

Thanks again for your susgestion and hope to received more for my new problem!