Abstract
Baculovirus infection of Sf9 cells at high densities, such as during mid- and late exponential phase, often results in a significant reduction of protein yield per cell, compared to the early exponential phase. Nutrient depletion has been considered as a major cause for the decreased protein yield. In this study, we report that the addition of nutrients (glucose, yeastolate ultrafiltrate, and lactalbumin hydrolysate) and small fraction of fresh medium at time of infection restores the expression level of actin and myosin V–HMM at late exponential phase (11.3 × 106 cells/ml) to that at early exponential phase (1.0 × 106 cells/ml). The relative yields of actin and myosin V–HMM were approximately equal at both phases (typically 200 mg of actin and 5 mg of myosin V–HMM per 1010 cells), i.e., the volumetric yield of proteins from the cell culture at late exponential phase was approximately tenfold higher than at early exponential phase. The functionality of the recombinant actin and myosin V–HMM was confirmed by measuring the rate of actin polymerization, actin-activated ATPase, and the gliding velocity of actin filaments in an in vitro motility assay.
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References
Bedard C, Tom R, Kamen A (1993) Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTI-EAA insect cell cultures: insights into growth limitation and metabolism. Biotechnol Prog 9(6):615–624. doi:10.1021/bp00024a008
Bedard C, Kamen A, Tom R, Massie B (1994) Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures. Cytotechnology 15(1–3):129–138
Elias CB, Zeiser A, Bedard C, Kamen AA (2000) Enhanced growth of Sf-9 cells to a maximum density of 5.2 × 10(7) cells per mL and production of beta-galactosidase at high cell density by fed batch culture. Biotechnol Bioeng 68(4):381–388
Ferrance JP, Goel A, Ataai MM (1993) Utilization of glucose and amino acids in insect cell cultures: quantifying the metabolic flows within the primary pathways and medium development. Biotechnol Bioeng 42(6):697–707. doi:10.1002/bit.260420604
Genzel Y, Ritter JB, Konig S, Alt R, Reichl U (2005) Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells. Biotechnol Prog 21(1):58–69. doi:10.1021/bp049827d
Hassell T, Butler M (1990) Adaptation to non-ammoniagenic medium and selective substrate feeding lead to enhanced yields in animal cell cultures. J Cell Sci 96(Pt 3):501–508
Hensler W, Singh V, Agathos SN (1994) Sf9 insect cell growth and beta-galactosidase production in serum and serum-free media. Ann NY Acad Sci 745:149–166
Kouyama T, Mihashi K (1981) Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur J Biochem/FEBS 114(1):33–38
Kubota H, Mikhailenko SV, Okabe H, Taguchi H, Ishiwata S (2009) D-loop of actin differently regulates the motor function of myosins II and V. J Biol Chem 284(50):35251–35258. doi:10.1074/jbc.M109.013565
Maranga L, Coroadinha AS, Carrondo MJ (2002) Insect cell culture medium supplementation with fetal bovine serum and bovine serum albumin: effects on baculovirus adsorption and infection kinetics. Biotechnol Prog 18(4):855–861. doi:10.1021/bp025514b
Nguyen B, Jarnagin K, Williams S, Chan H, Barnett J (1993) Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system. J Biotechnol 31(2):205–217
Ohki T, Mikhailenko SV, Morales MF, Onishi H, Mochizuki N (2004) Transmission of force and displacement within the myosin molecule. Biochemistry 43(43):13707–13714. doi:10.1021/bi048954f
Ohki T, Ohno C, Oyama K, Mikhailenko SV, Ishiwata S (2009) Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin. Biochem Biophys Res Commun 383(1):146–150. doi:10.1016/j.bbrc.2009.03.144
Pullman ME, Penefsky HS, Datta A, Racker E (1960) Partial resolution of the enzymes catalyzing oxidative phosphorylation. I. Purification and properties of soluble dinitrophenol-stimulated adenosine triphosphatase. J Biol Chem 235:3322–3329
Reuveny S, Kim YJ, Kemp CW, Shiloach J (1993) Production of recombinant proteins in high-density insect cell cultures. Biotechnol Bioeng 42(2):235–239. doi:10.1002/bit.260420211
Wang MY, Kwong S, Bentley WE (1993) Effects of oxygen/glucose/glutamine feeding on insect cell baculovirus protein expression: a study on epoxide hydrolase production. Biotechnol Prog 9(4):355–361. doi:10.1021/bp00022a002
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This work was supported by the Grants-in-Aid for Specially Promoted Research and Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.I.
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Ohki, T., Mikhailenko, S.V., Arai, T. et al. Improvement of the yields of recombinant actin and myosin V–HMM in the insect cell/baculovirus system by the addition of nutrients to the high-density cell culture. J Muscle Res Cell Motil 33, 351–358 (2012). https://doi.org/10.1007/s10974-012-9323-8
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DOI: https://doi.org/10.1007/s10974-012-9323-8