Reductive methylation of amino groups (lysines and the N-terminus) is a fairly routine chemical modification that should drop the solubility of your protein. An easy-to-use protocol can be found in Walter et al (2007) "Lysine Methylation as a Routine Rescue Strategy for Protein Crystallization." Structure, 14:1617-1622
Cheers, Stephen On 8/7/07, [EMAIL PROTECTED] <[EMAIL PROTECTED]> wrote: > 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 > > > -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
