Title: Studies of the intermediary metabolism in cultured cells of the insect Spodoptera frugiperda using 13C- or 15N-labelled tracers
Abstract: Abstract Background Insect cells can serve as host systems for the recombinant expression of eukaryotic proteins. Using this platform, the controlled expression of 15 N/ 13 C labelled proteins requires the analysis of incorporation paths and rates of isotope-labelled precursors present in the medium into amino acids. For this purpose, Spodoptera frugiperda cells were grown in a complex medium containing [U- 13 C 6 ]glucose. In a second experiment, cultures of S. frugiperda were grown in the presence of 15 N-phenylalanine. Results Quantitative NMR analysis showed incorporation of the proffered [U- 13 C 6 ]glucose into the ribose moiety of ribonucleosides (40 – 45%) and into the amino acids, alanine (41%), glutamic acid/glutamine (C-4 and C-5, 30%) and aspartate/asparagine (15%). Other amino acids and the purine ring of nucleosides were not formed from exogenous glucose in significant amounts (> 5%). Prior to the incorporation into protein the proffered 15 N-phenylalanine lost about 70% of its label by transamination and the labelled compound was not converted into tyrosine to a significant extent. Conclusion Growth of S. frugiperda cells in the presence of [U- 13 C 6 ]glucose is conducive to the fractional labelling of ribonucleosides, alanine, glutamic acid/glutamine and aspartic acid/asparagine. The isotopolog compositions of the ribonucleosides and of alanine indicate considerable recycling of carbohydrate intermediates in the reductive branch of the pentose phosphate pathway. The incorporation of 15 N-labelled amino acids may be hampered by loss of the 15 N-label by transamination.