Abstract
The methylotrophic yeast Pichia pastoris is now an established expression system for the production of recombinant protein for nuclear magnetic resonance (NMR) studies. It is capable of expressing high levels of heterologous proteins and possesses the ability to perform many of the posttranslational modifications of higher eukaryotes. Here, we describe efficient methods for the production of uniformly 13C,15N-labeled proteins in shake flasks and of uniformly 13C,15N-labeled and 2H,13C,15N-labeled proteins in fermenters. We also provide details of two chromatographic procedures, cation exchange and concanavalin A lectin affinity, that have proved useful in purifying P. pastoris-expressed proteins for NMR studies.
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References
Higgins, D. R. and Cregg, J. M. (1998) Introduction to Pichia pastoris. In Methods in Molecular Biology, vol. 103: Pichia Protocols (Higgins, D. R. and Cregg, J. M., eds.), Humana, Totowa, NJ, pp. 1–15.
Cregg, J. M., Shen, S., Johnson, M., and Waterham, H. R. (1998) Classical genetic manipulation. In Methods in Molecular Biology, vol. 103: Pichia Protocols (Higgins, D. R. and Cregg, J. M., eds.), Humana, Totowa, NJ, pp. 17–26.
Lin Cereghino, G. P., Lin Cereghino, J., Ilgen, C., and Cregg, J. M. (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr. Opin. Biotechnol. 13, 1–4.
Grinna, L. S. and Tschopp, J. F. (1989) Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris. Yeast 5, 107–115.
Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology 11, 905–910.
Laroche, Y., Storme, V., De Meutter, J., Messens, J., and Lauwereys, M. (1994) High level secretion and very efficient isotopic labelling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast Pichia pastoris. Biotechnology 12, 1119–1124.
Cregg, J. M., Barringer, K. J., Hessler, A. Y., and Madden, K. R. (1985) Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 5, 3376–3385.
Scorer, C.A., Clare, J. J., McCombie, W. R., Romanos, M. A., and Sreekrishna, K. (1994) Rapid selection using G418 of high-copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology 12, 181–184.
Romanos, M. A. (1995) Advances in the use of Pichia pastoris for high-level gene expression. Curr. Opin. Biotechnol. 6, 527–533.
Invitrogen Corporation. Pichia Expression System: Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris, Version G. Available at http://www.invitrogen.com/content/sfs/manuals/pich_man.pdf. Accessed 03/01/04.
Stratton, J., Chriuvolu, V., and Meagher, M. (1998) High cell-density fermentation. In Methods in Molecular Biology, vol. 103: Pichia Protocols (Higgins, D. R. and Cregg, J. M., eds.), Humana, Totowa, NJ, pp. 107–120.
Invitrogen Corporation. Pichia Fermentation Process Guidelines. Available at http://www.invitrogen.com/content/sfs/manuals/pichiaferm_prot.pdf. Accessed 03/01/04.
Clare, J. J., Romanos, M. A., Rayment, F. B., Rowedder, J. E., Smith, M. A., Payne, M. M., et al. (1991) Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105, 205–212.
Gleeson, M. A. G., White, C. E., Meininger, D. P., and Komives, E. A. (1998) Generation of protease-deficient strains and their use in heterologous protein expression. In Methods in Molecular Biology, vol. 103: Pichia Protocols (Higgins, D. R. and Cregg, J. M., eds.), Humana, Totowa, NJ, pp. 81–94.
Bright, J. R., Pickford, A. R., Potts, J. R., and Campbell, I. D. (1999) Preparation of isotopically labelled recombinant fragments of fibronectin for functional and structural study by heteronuclear nuclear magnetic resonance spectroscopy. In Methods in Molecular Biology, vol. 139: Extracellular Matrix Protocols (Streuli, C. and Grant, M., eds.), Humana, Totowa, NJ, pp. 59–69.
Cremata, J. A., Montesino, R., Quintero, O., and Garcia, R. (1998) Glycosylation profiling of heterologous proteins. In Methods in Molecular Biology, vol. 103 Pichia Protocols (Higgins, D. R. and Cregg, J. M., eds.), Humana, Totowa, NJ, pp. 95–105.
Brady, C. P., Shimp, R. L., Miles, A. P., Whitmore, M., and Stowers, A. W. (2001) High-level production and purification of P30P2MSP119, an important vaccine antigen for malaria, expressed in the methylotrophic yeast Pichia pastoris. Protein Expr. Purif. 23, 468–475.
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Pickford, A.R., O’Leary, J.M. (2004). Isotopic Labeling of Recombinant Proteins from the Methylotrophic Yeast Pichia pastoris . In: Downing, A.K. (eds) Protein NMR Techniques. Methods in Molecular Biology™, vol 278. Humana Press. https://doi.org/10.1385/1-59259-809-9:017
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DOI: https://doi.org/10.1385/1-59259-809-9:017
Publisher Name: Humana Press
Print ISBN: 978-1-58829-246-9
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