Characterization of yeastolate fractions that promote insect cell growth and recombinant protein production
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Yeastolate is effective in promoting growth of insect cell and enhancing production of recombinant protein, thus it is a key component in formulating serum-free medium for insect cell culture. However, yeastolate is a complex mixture and identification of the constituents responsible for cell growth promotion has not yet been achieved. This study used sequential ethanol precipitation to fractionate yeastolate ultrafiltrate (YUF) into six fractions (F1–F6). Fractions were characterized and evaluated for their growth promoting activities. Fraction F1 was obtained by 65% ethanol precipitation. When supplemented to IPL-41 medium at a concentration of 1 g L−1, fraction F1 showed 71% Sf-9 cell growth improvement and 22% β-galactosidase production enhancement over YUF (at 1 g L−1 in IPL-41 medium). However, the superiority of F1 over YUF on promoting cell growth gradually diminished as its concentration in IPL-41 medium increased. At 4 g L−1, the relative activity of F1 was 93% whereas YUF was 100% at the same concentration. At 1 g L−1, four other fractions (F2–F5) precipitated with higher ethanol concentrations and F6, the final supernatant, showed growth promoting activities ranging from 32 to 80% as compared to YUF (100%). Interestingly, a synergistic effect on promoting cell growth was observed when F6 was supplemented in IPL-41 medium in presence of high concentrations of F1 (>3 g L−1). The results suggest that ethanol precipitation was a practical method to fractionate growth-promoting components from YUF, but more than one components contributed to the optimum growth of Sf-9 cells. Further fractionation, isolation and identification of individual active components would be needed to better understand the role of these components on the cell metabolism.
KeywordsInsect cell Yeastolate Serum-free medium
The authors wish to thank Louis Bisson and Alice Bernier for providing assistance in analysis of amino acids and size exclusion chromatography. The authors also appreciated helpful discussions with Cynthia Elias and Faustinus Yeboah.
- Bridson EY, Brecker A (1970) Design and formulation of microbial culture media. In: Methods in microbiology, vol. 3A. Academic Press, pp 252–256Google Scholar
- Burteau CC, Verhoeye FR, Mols JF, Ballez JS, Agathos SN, Schneider YJ (2003) Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-gamma-producing CHO cell line. In vitro Cell Dev Biol Anim 39:291–296PubMedCrossRefGoogle Scholar
- Heidemann R, Zhang C, Qi H, Rule JL, Rozales C, Park S, Chuppa S, Ray M, Micheals J, Konstantin K, Naveh D (2000) The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnol 32:157–167CrossRefGoogle Scholar
- Kelly M (1983) Yeast extract. In: Godfrey T, Reichelt M (eds) Industrial enzymology, the application of enzymes in industry. Nature press, New York, pp 457–464Google Scholar
- Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G, Wang DIC (1999) Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium. Biotechnol Bioeng 62:423–335Google Scholar
- Peptone Technical Manual, BD Biopharmaceutical Production, BD Biosciences, Sparks, MD, USA. May 2002Google Scholar
- Scopes RK (1987) Protein purification, 2nd edn. Chapter 3: separation by precipitation. Springer-Verlag, NYGoogle Scholar
- Sommer R (1996) Yeast extracts: production, properties and components. In: 9th International Symposium on Yeasts, Sydney, August 1996Google Scholar
- Taylor KACC (1995) A modification of the phenol/sulfuric acid assay for total carbohydrates giving more comparable absorbances. Appl Biochem Biotechnol 53:207–214Google Scholar