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protein expression problem - (Sep/05/2008 )

[attachment=5259:codon_usage.jpg]Hi, I have cloned a 75 AA protien (2 disulfide bonds) into the pET16b expression vector, and checked the sequence by sequencing. I've tried to induction in BL21(DE3) and BL21(DE3)plysS with different concentration of IPTG (0.1, 0.5, 1mM), but had very low or no expression. As my SDS-PAGE shows that there is no difference among the soluble fraction between the control and induced. For the insoluble fraction, there is a slightly stronger band at the expected size in the induced than the control. I don't have the reagents to do a western.

My culture condition is 37C and IPTG was added at OD600=0.65 for another 3 hours before harvesting, and the culture medium is LB.
I've read that induction at a smaller OD600 (0.3) and lower temperature for prolonged time helps. Also people some time use M9 medium. I don't have much experience with this. How much difference would culture condition make in terms of expression?

I also checked the codon usage, 10 of the codons have frequency less than 15%, Rosseta can fix most of these. How often do you see codon usage pose a problem?

Thank you very much for your advice.


problem solved by Rosetta2(DE3)plyss