An improved protocol for amino acid type-selective isotope labeling in insect cells
| Autor: | William J. Metzler, Luciano Mueller, Yaqun Zhang, Mian Gao, Nicolas Szapiel, Mark R. Witmer, David Hedin, Deepa Calambur, Dianlin Xie, Hui Wei, Frank Marsilio, Andrew E Douglas, Ping Zhang |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Spodoptera Biochemistry 03 medical and health sciences chemistry.chemical_compound Stable isotope labeling by amino acids in cell culture Sf9 Cells Animals Amino Acids Nuclear Magnetic Resonance Biomolecular Spectroscopy chemistry.chemical_classification Carbon Isotopes Growth medium Chromatography Nitrogen Isotopes 030102 biochemistry & molecular biology Insect cell culture Yeast Culture Media Amino acid 030104 developmental biology chemistry Focal Adhesion Kinase 1 Isotope Labeling Protein Biosynthesis Reagent Yield (chemistry) CD4 Antigens Fermentation Baculoviridae |
| Zdroj: | Journal of Biomolecular NMR. 68:237-247 |
| ISSN: | 1573-5001 0925-2738 |
| Popis: | An improved expression protocol is proposed for amino acid type-specific [13C], [15N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449-456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents. |
| Databáze: | OpenAIRE |
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