To obtain soluble proteins, a key step is to select a suitable expression cells depending on the original source of the recombinant protein. One of the primary reasons for misfolding and subsequent aggregation is some proteins require post translational modifications to correctly fold and remain soluble. Ideally, the protein expression system chosen should match the source of protein. So if making recombinant bacterial protein, uses a bacterial system, for mammalian, use a mammalian system, etc. Unfortunately, this is not a simple:
The main drawback of recombinant protein production in prokaryotic (E. coli) expression system is the lack of post translational modifications. E. coli expression system offer more protein in less time and many proteins do not require post translational modifications for their solubility and activity, so these are the preferred method for expression. But what if your protein is targeted to inclusion bodies?
The manipulation of the growth temperature of bacteria can play a key role in obtaining soluble proteins. Typically bacteria are grown at 37°C for optimal E.coli growth, but slowing the cell growth during protein induction is advisable to increase the amount of soluble protein. Recommended temperatures between 16-25°C will allow the cells to grow at a slower rate and help prevent proteins aggregating and being targeted to inclusion bodies.
An additional step of cold shock has been shown to increase the chances of obtaining soluble protein. Cold shock involves incubating the culture for 1hr at 4°C or on ice prior adding the inducer. The cold shock treatment induces the production of a group of 13 cold shock proteins, including CspA . These cold shock proteins help the organism to survive the stress of cold shock. One feature of this survival is to slow down protein production and this, in turn, produces less chance of inclusion body formation. Cold shock proteins are known to have a short half-life of around 12 seconds at 37°C and their half-life increases to more than 20 minutes when cells are subjected to temperatures below 10°C .