Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation
| Autor: | M J, Wood, E A, Komives |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Glycerol
Carbon Isotopes Protein Folding Glycosylation Magnetic Resonance Spectroscopy Epidermal Growth Factor Nitrogen Isotopes Nitrogen Protein Conformation Thrombomodulin Molecular Sequence Data Temperature Mycology Carbon Peptide Fragments Pichia Recombinant Proteins Culture Media Protein Structure Tertiary Glucose Ammonium Sulfate Isotope Labeling Fermentation Amino Acid Sequence Protein Processing Post-Translational |
| Zdroj: | Journal of biomolecular NMR. 13(2) |
| ISSN: | 0925-2738 |
| Popis: | Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P. pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P. pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P. pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10-100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N-labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment. |
| Databáze: | OpenAIRE |
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