September 1, 2009 23:18
Succeeding with what you have is a key characteristic of successful researchers and their projects. Applied to protein crystallography this may involve using materials that are usually deemed useless. Take for example proteins that express in E.coli as inclusion bodies rather than in soluble form. At this point in the game you can either go back to creating new expression clones or make use of the protein available. There's often a lot of material in inclusion bodies and the first purification step, differential centrifugation already yields enriched samples. While that's good news, the trouble is the need to refold the protein into its native form prior to setting up crystallization trials.
I like the concept of on-column refolding as described in J. Struct Funct. Genomics. 2005; 6(2-3): 177-182. A primer is available here.
There Natalia et. al. describe on-column protein refolding specifically for protein crystallization. His-tagged proteins in inclusion bodies are solubilized in Urea or Guanidinium Hydrochloride and bound to a Ni-affinity resin. They wash the bound protein with detergent (Triton X-100 - makes me cringe, but it does the job) and remove it completely with beta-cyclodextrin prior to elution with imidazole.
This method is based on a procedure developed in Sam Gellman's lab (D. Rozema and S.H. Gellman, J. Biol. Chem. 271(7), 3478-3487 (1996). Since the detergent aids the renaturation process in ways similar to chaperones in vivo the term 'artificial chaperone-assisted refolding' was coined for this approach.
I don't know how many crystals and structures this method has produced other than the examples mentioned in the paper. But this on-column, artificial chaperone-assisted refolding is quick and should be simple enough to try before going back to the drawing board.