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Dear cc4bb enthusiasts, This is slightly off topic but many protein crystallographers might be familiar with this problem. I have been trying to over-express a bacterial (non-E.Coli) protein in E.Coli and more than 80% goest to inclusion bodies. I tried the following Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5 mM) Cold shock for 30 minutes in ice before induction Slow rotation speed at 18 degrees O/N after induction While these steps helped a bit I still get about 50-60% of my protein in inclusion bodies. I would like to know what other steps do you suggest to enhance the yield in the soluble fraction (without changing the host strain or manipulating the DNA) Thanks in advance Ivan