Insights into the central metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5) insect cells by radiolabeling studies.
The insect cell baculovirus expression vector system (BEVS) is one of the most commonly used expression systems for recombinant protein production. This system is also widely used for the production of recombinant virus and virus-like particles. Although several published reports exist on recombinant protein expression using insect cells, information dealing with their metabolism in vitro is relatively scarce. In this work we have analyzed the metabolism of glucose and glutamine, the main carbon and/or energy compounds, of the two most commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5). Radiolabeled substrates have been used to determine the flux of glucose carbon entering the tricarboxylic acid cycle (TCA) and the pentose phosphate (PP) pathway by direct measurement of 14CO2 produced. The percentage of total glucose metabolized to CO2 via the TCA cycle was higher in the case of the Sf-9 (2.7%) compared to Tn-5 (0.6%) cells, while the percentage of glucose that is metabolized via the PP pathway was comparable at 14% and 16% for the two cell lines, respectively. For both cell lines, the remaining 83% of glucose is metabolized through other pathways generating, for example, lactate, alanine, etc. The percentage of glutamine oxidized in the TCA cycle was approximately 5-fold higher in the case of the Tn-5 (26.1%) as compared to the Sf-9 cells (4.6%). Furthermore, the changes in the metabolic fluxes of glucose and glutamine in Tn-5-PYC cells, which have been engineered to express a cytosolic pyruvate carboxylase, have been studied and compared to the unmodified cells Tn-5. As a result of this metabolic engineering, significant increase in the percentage of glucose oxidized in the TCA cycle (3.2%) as well as in the flux through the PP pathway (34%) of the Tn-5-PYC were observed.
Benslimane
Chouki
C
Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montréal, Québec, Canada H4P 2R.
Elias
Cynthia B
CB
Hawari
Jalal
J
Kamen
Amine
A
D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D002247:Carbon Isotopes; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002952:Citric Acid Cycle / Q000502:physiology; D003470:Culture Media; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D009036:Moths / Q000166:cytology / Q000378:metabolism*; D011766:Pyruvate Carboxylase / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors
D002247:Carbon Isotopes; D003470:Culture Media; D011994:Recombinant Proteins; D005973:Glutamine; D002245:Carbon Dioxide; D011766:Pyruvate Carboxylase; D005947:Glucose