A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells.
Whereas bacterial expression systems are widely used for production of uniformly or selectively (15)N-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 (15)N-labeled proteins in insect cells. The quantities of (15)N-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 (15)N-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.
Brüggert
Michael
M
Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany.
Rehm
Till
T
Shanker
Sreejesh
S
Georgescu
Julia
J
Holak
Tad A
TA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D018411:Spodoptera / Q000166:cytology / Q000235:genetics*
D000596:Amino Acids; D003470:Culture Media; D009587:Nitrogen Isotopes; D011506:Proteins; D005982:Glutathione Transferase