Optimization of buffer solutions for protein crystallization.
Increasing the solubility of protein stock solutions to above that in a standard chromatography buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl) led to an increase in the number of crystallization conditions for ten globular proteins subjected to two crystal screens: the Index and Precipitant/Precipitant-Additive (P/PA) Screens. Solubility enhancement of protein stock solutions was achieved through screening and selection of buffer components to formulate an optimal buffer. Relative improvements in solubility were estimated through protection against the precipitation of protein by polyethylene glycol 8000. Proteins with limited solubility improvement in optimal buffer showed an enhancement in solubility on addition of glycerol. Maximum solubility was then determined by the concentration of optimized solutions until precipitate formed. The supernatant concentration then provided an estimate of the upper limit of protein solubility. This 'solubility' estimate is used to specify the initial concentration of the protein used in the screening experiments and is an important step in successful crystallization. Buffer optimization and establishment of initial protein concentration for crystal screening based on solubility estimates provides a methodology for improved crystal screening results.
Gosavi
Rajendrakumar A
RA
Department of Chemical Engineering, The University of Toledo, Toledo, OH 43606, USA.
Mueser
Timothy C
TC
Schall
Constance A
CA
D002021:Buffers; D003460:Crystallization / Q000379:methods; D011506:Proteins / Q000737:chemistry*; D012995:Solubility; D012996:Solutions / Q000737:chemistry*
D002021:Buffers; D011506:Proteins; D012996:Solutions