Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics
- 226 Downloads
We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.
Unable to display preview. Download preview PDF.
- Quevillon-Cheruel, S., Collinet, B., Zhou, C.Z., Minard, P., Blondeau, K., Henkes, G., Aufrere, R., Coutant, J., Guittet, E., Lewit-Bentley, A., Leulliot, N., Ascone, I., Sorel, I., Savarin, P., de La Sierra Gallay, I.L., de la Torre, F., Poupon, A., Fourme, R., Janin, J. and van Tilbeurgh, H. (2003) J. Synchrotron Radiat. 10, 4–8.CrossRefPubMedGoogle Scholar
- Stratton, J., Chiruvolu, V. and Meagher, M. (1998) In Methods in Molecular Biology, Vol. 103: Pichia Protocols (Eds., Higgins, D.R. and Cregg, J.M.), Humana Press, Totowa, NJ, pp. 107–120.Google Scholar