Re: [ccp4bb]: Skin formation in crystallization trials:

[james whisstock]

Could you design your constructs so that you could cleave off the MBP using TeV or another specific protease (or have you already done so?), or use a His rather than an MBP tag if solubility is not a problem? Do you have evidence that the individual domains are folded and functional when expressed with the MBP?  Generally large tags with flexbile linkers hamper crystal growth - there is a review by DR Smyth et al., 2003 Prot Sci which details a few successful examples though where rigid linkers with MBP were used. We have had several examples where crystals have formed under a thick skin - plucking the skin off with a loop or spike followed by rapid harvesting of the crystals that were'nt stuck to the skin worked for us. J [EMAIL PROTECTED] wrote: I am working on a protein with three domains. I have purified three different constructs – only one domain or two domains or all the three domains. All these proteins, I expressed as MBP fusion and purified well and runs as monomer in gel-filtration. Surprising all these proteins form skin in almost all conditions (PEG or salt) of Nextal and Hampton crystallization screen. These domains have some cysteines,and ddition of reducing agent (DTT or TCEP) during crystallization trials did not help and I still get skins. The proteins run as monomer in gel-filtration column even with out any reducing agents. Searching in Web did not give me clear answer. Any body has any such experience/suggestions.   Thanks muthu