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Low expression? and reduction in OD600 in multiple strains

protein expression host strains

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#1 Missle

Missle

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Posted 12 April 2013 - 08:08 AM

I've been trying to express a couple of different proteins using a T7-based expression vector in E.coli. They both are predicted to be good candidates for bacterial expression (size, RaCC, hydrophobicity, etc...) I've looked (in parallel) at BL21 (DE3), Rosetta 2 (DE3), and BL21 (DE3) pLysS. I have 3 samples I'm analyzing: non-induced, induced w/0.05mM IPTG, and induced w/ 0.5mM IPTG. I grow at 37C until an OD600 of 0.5 to 1.0 and then induce overnight at 18C. I haven't acheived good expression for either target but what is bothering me is the trend I'm seeing with the three cell lines. For both proteins, the OD600 at harvest shows very little reduction between induced and non-induced in the BL21 & pLysS strains but good 50% + reduction with the Rosetta which should indicate that the Rosetta strain is expressing my protein (shouldn't it?) but if it is I can't tell. I don't see it in the insoluble or soluble fraction. At first I thought it was toxicity but if that were the case, shouldn't BL21 look more like Rosetta interms of OD600 reduction?

I appreciate any ideas!





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