Inclusion body formation all or nothing? - (Sep/01/2006 )
In inclusion body formation (for example in overexpressing a protein), is the process all or nothing? For example, if I over express protein X and it reahces beyond 0.1mg/mL or something, does most of it automatically become inclusion bodies and there is no point in inducing anymore expression? On the other hand, can I keep over expressing it and then increase the soluble fraction as well as the inclusion body fraction (to harvest more soluble fraction of course!)?
Any experience or references to information on this would be appreciated, thanks.
well, i think that inclusion body formation is not always due to concentration but also to the rate at which the protein is formed. if it is being expressed so rapidly that theres no time for it to fold properly, its hydrophobic patches get aggregated with other protein molecules patches and they form an insoluble mass. you can increase solubility by expressing at lower temperatures (16 degrees, etc) which slows the kinetics of the whole works and you are far more likely to get soluble protein then.
That may be a good idea, I've heard of that one too. I have also heard sometimes that increasing temperature may change the ratio of inclusion body protein to soluble protein, perhaps by heat shock proteins. Maybe the best way to go is to try several conditions. I was mainly wondering, though, if perhaps I continued to over express a protein even after inclusion body formation it might reach a "maximum rate" of formation of the inclusion state and produce more soluble protein as well??? I'm not sure.
I was also going to try waiting until the culture was very dense (large OD650) before induction to help increase yield a bit. How much does that usually help or are there problems associated with that strategy as well?