J. Struct. Funct. Genomics
Small-scale, semi-automated purification of eukaryotic proteins for structure determination.
Frederick, R.O., Bergeman, L., Blommel, P.G., Bailey, L.J., McCoy, J.G., Song, J., Meske, L., Bingman, C.A., Riters, M., Dillon, N.A., Kunert, J., Yoon, J.W., Lim, A., Cassidy, M., Bunge, J., Aceti, D.J., Primm, J.G., Markley, J.L., Phillips, G.N. Jr. and Fox, B.G.
Notes: These authors describe a simple, small-scale screening method for recombinant polyhistidine-tagged proteins. They used the Maxwell® 16 Polyhistidine Protein Purification Kit and Maxwell® 16 Instrument to purify the proteins, and characterized the purified proteins by NMR and X-Ray analysis. They first used the Flexi® Vector System to clone the genes of interest into expression vectors containing either an N-terminal TEV protease cleavable His8-Maltose Binding Protein (His8-MBP) tag, or an in vivo cleaved His8-MBP tag. For small-scale screening, E. coli expressing fusion proteins were grown for 24 hours in auto-induction medium in 96-well growth blocks, harvested by centrifugation and resuspended to an OD600 of 20 in 1ml of 50mM HEPES (pH 7.5) containing a protease inhibitor cocktail. Aliquots of these cells were then added directly to the Maxwell® kit cartridge, and automated purification performed. The purified proteins were analyzed by SDS-PAGE or using a Caliper LC90 electrophoresis system. For purification of proteins for use in NMR or crystallography studies, 50ml overnight cultures were harvested and resuspended in 10 ml of 50mM HEPES (pH 7.5) containing 10 units of benzonase, 1mg/ml lysozyme, and protease inhibitor cocktail. The cell suspension was sonicated, and aliquots were then added to the Maxwell® 16 cartridge for purification. The authors purified 14 different proteins from humans, frogs, and zebrafish using this method. Detailed reports of yields obtained and subsequent analyses are provided in the paper. (3799)
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