The key role of glutamine for protein expression and isotopic labeling in insect cells.

Nuclear magnetic resonance studies of many physiologically important proteins have long been impeded by the necessity to express such proteins in isotope-labeled form in higher eukaryotic cells and the concomitant high costs of providing isotopelabeled amino acids in the growth medium. Economical routes use isotope-labeled yeast or algae extracts but still require expensive isotope-labeled glutamine. Here, we have systematically quantified the effect of 15N2-glutamine on the expression and isotope labeling of different proteins in insect cells. Sufficient levels of glutamine in the medium increase the protein expression by four to five times relative to deprived conditions. 1H-15N nuclear magnetic resonance spectroscopy shows that the 15N atoms from 15N2-glutamine are scrambled with surprisingly high (60-70%) efficiency into the three amino acids alanine, aspartate, and glutamate. This phenomenon gives direct evidence that the high energy demand of insect cells during baculovirus infection and concomitant heterologous protein expression is predominantly satisfied by glutamine feeding the tricarboxylic acid cycle. To overcome the high costs of supplementing isotope-labeled glutamine, we have developed a robust method for the large-scale synthesis of 15N2-glutamine and partially deuterated 15N2-glutamine-a,ß,ß-d3 from inexpensive precursors. An application is shown for the effective large-scale expression of the isotope-labeled ß1-adrenergic receptor using the synthesized 15N2-glutamine-a,ß,ß-d3. [ABSTRACT FROM AUTHOR]