To optimize protein expression in SHuffle strains, we recommend that you first determine the best SHuffle expression strain for your protein.
Currently, there are two SHuffle cell lines: SHuffle based on E.coli K12, and SHuffle Express based on E.coli B. Optimum expression of a protein may depend on the SHuffle expression strain used. We therefore recommend trying both SHuffle T7 competent E.coli and SHuffle T7 Express competent E.coli, and picking the cell line that expresses your protein better.
If T7 expression is not necessary, compare SHuffle T7 competent E.coli to SHuffle Express competent E.coli. If T7 driven expression of a protein is toxic, switch to a non leaky lysY version.
The next step should be to optimize expression temperatures. We recommend trying lower expression temperatures, as lower temperatures can lead to higher yields of correctly folded protein.
Further optimization of protein expression conditions can be accomplished by testing various inducer concentrations, and trying auto expression.
Finally, even further optimization of expression conditions can be achieved by testing the co-expression of various chaperones and folding factors, using solubility tags, and even adding redox active molecules to the growth or lysis media.
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