membrane protein crystallization

Leigh Ann Lipscomb lipscomb at linus.bmb.uga.edu
Wed Feb 9 10:48:56 EST 2000


We are experiencing problems with crystallization of an integral
membrane protein.  We are working on the protein-ATP complex.  I am
writing to ask for advice.  Our protein is soluble to 16 mg/ml in 25 mM
Tris pH 8.0, 25 mM NaCl, in the presence of 0.6% C16H34O5 (detergent,
also called C8E4).  Functional assays have indicated that it is active
under these conditions.  For the Hampton Crystal Screen I array, we got
some small crystals under conditions 5, 21, and 26 (dimensions no larger
than 0.05 mm on any edge).  We had the most success in optimization
(i.e. the biggest crystals) with condition #21.  At the  moment the best
conditions we have arrived at have 0.2M ammonium acetate, 0.1 M sodium
citrate pH 5.1, 24% MPD in the well solution.  The crystals are large in
2 dimensions 0.2 mm or larger in cases, yet very small in the third
dimension (0.05 mm).  They diffract poorly; different crystals give
diffraction to between 10 and 6 Angstrom resolution.   We have tried the
detergent screen, as well as Additive Screen 1 from Hampton, and we have
tested the effects of temperature.  The ratio of ATP to protein is 5 to
1.  We have removed the N-terminal tag from the subcloning experiment.
The protein is very pure by every assay I can think of (silver stained
SDS, for example).  Does anyone have advice?

Thanks very much.

Leigh Ann Lipscomb








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