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A novel medium for expression of proteins selectively labeled with N-15-amino acids in Spodoptera frugiperda (Sf9) insect cells

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons77819

Brüggert,  M.
Holak, Tad / NMR Spectroscopy, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78552

Rehm,  T.
Huber, Robert / Structure Research, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78697

Shanker,  S.
Holak, Tad / NMR Spectroscopy, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78000

Georgescu,  J.
Holak, Tad / NMR Spectroscopy, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons78123

Holak,  T. A.
Holak, Tad / NMR Spectroscopy, Max Planck Institute of Biochemistry, Max Planck Society;

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Zitation

Brüggert, M., Rehm, T., Shanker, S., Georgescu, J., & Holak, T. A. (2003). A novel medium for expression of proteins selectively labeled with N-15-amino acids in Spodoptera frugiperda (Sf9) insect cells. Journal of Biomolecular NMR, 25(4), 335-348.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0010-6C47-2
Zusammenfassung
Whereas bacterial expression systems are widely used for production of uniformly or selectively N-15-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively N-15-labeled proteins in insect cells. The quantities of N-15-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the N-15-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.