Influence of targeted asparagine starvation on extra- and intracellular amino acid pools of cultivated Chinese hamster ovary cells.
For the development of an expression system with an amino-acid-inducible promoter, the influence of extracellular stress, by starvation of the non-essential amino acid asparagine, on the extra- and intracellular amino acid pool was investigated. Therefore a widely used nontransformed CHO cell line was cultivated in a serum-free and optimized DMEM/F12 medium in repeated batch mode. During the last repeat the medium contained no asparagine. The cells could compensate totally for this lack by an increased conversion of aspartate, glutamate, asparagine, serine, glutamine and arginine, while almost the whole intracellular pool of amino acids decreased. By this enhanced metabolic activity the maximum growth rate rose from 0.8 day-1 in complete medium to 1.1 day-1 in asparagine-free medium. The exceptional increase in asparagine biosynthesis points to a strong activation of asparagine synthetase, the key enzyme within the asparagine biosynthesis pathway. The regulation mechanism for the asparagine synthetase at the transcription level had to be analysed further in detail and will lead to an asparagine-sensitive promotor. To investigate reaction cascades that influence the protein synthesis or the overall gene expression, one had to look carefully at intracellular amino acid levels, because of their importance for polypeptide synthesis and energy supply, but also because of their obvious sensitivity to extracellular stresses.
Seewöster
T
T
Institute of Cell Culture Technology, Faculty of Technical Sciences, University of Bielefeld, Germany.
Lehmann
J
J
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001216:Asparagine / Q000172:deficiency*; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects
D000596:Amino Acids; D016895:Culture Media, Serum-Free; D001216:Asparagine