Yvonne, Several 'old' proteins have been crystallized from insanely high concentraitons - concanavalin A for instance can be grown from 100-250 mg/ml solutions by means of 'salting in' using microdialysis. This is of course highly labor-intensive and also expensive on the protein side.
Look at the distribution of charged residues on the protein (especially helpful if you have a model of some sort). I would recommend mutating some of them to non-charged or even nonpolar residues in order to lower solubility. Alternatively, you could try preparing chemical derivatives of the protein using e.g. heavy atoms or amine-modifying reagents like NHS. If you block enough amines, your solubility should go down and your pI will change as well. Likewise you could try modifying exposed acidic residues etc. Mutagenesis can in the end be easier to do because chemical modification tends to produce complex mixtures of products, unless you do it with a huge excess of the reagent and allow the reaction to proceed to exhaustion. Artem > Our lab is trying to crystallize a highly soluble (100+ mg/ml) protein > with a molecular weight of 35 kd. > > The protein was screened against 1536 conditions at 20 mg/mL. Most drops > were either clear or produced "bubbles" (often oily looking). The few > that had precipitate contained high concentrations of K3PO4, cobalt, or > zinc. We have tried repeating some of the bubble conditions at 100+ > mg/mL and are still getting clear drops or bubbles. > > Is there something about highly soluble proteins and/or secreted > proteins and/or proteins with unusual portions of their sequence that > needs to be considered in order to successfully crystallize it? > > I am considering trying "salting out" using dialysis, and also adding > ligands/inhibitors. The protein is in 50 mM NaCl plus 50 mM buffer. > > I welcome thoughts and suggestions on crystallization ideas, > publications, etc. > > Thank you > > Yvonne >
