J Bacteriol Virol.  2012 Mar;42(1):69-75. 10.4167/jbv.2012.42.1.69.

Enhancement of Protein Productivity of Recombinant Hepatitis A Virus VP1 in Stably Transfected Drosophila melanogaster S2 Cells

Affiliations
  • 1Graduate School of Biotechnology, Kyung Hee University, Yongin, Korea. ischung@khu.ac.kr
  • 2Medican Co., Ltd., Seoul, Korea.
  • 3Korea Food Research Institute, Seongnam-si, Kyunggi-do, Korea.
  • 4Department of Microbiology & Research Institute for Translational System Biomics, Chung-Ang University College of Medicine, Seoul, Korea.

Abstract

The effect of DMSO and sodium butyrate on the production of recombinant hepatitis A virus (HAV) capsid protein VP1 was evaluated and optimized in the culture of stably transfected Drosophila melanogaster S2 cells using culture plates and spinner flasks. The effect of DMSO and sodium butyrate was also evaluated to improve the recombinant VP1 production in stably transfected Drosophila S2 cells. A production level of 0.88 mg of recombinant VP1/liter was obtained in the culture-plate culture of stably transfected S2 cells at 6 days after induction with 0.5 mM CuSO4. The supplements of 2% DMSO and 10 mM sodium butyrate at 4 days post-inoculation increased recombinant VP1 accumulation by 141 and 104%, respectively, resulting in 2.17 and 1.7 mg/liter of recombinant VP1 production. In spinner flasks, recombinant VP1 production reached maximum level at 9 days after induction with 0.5 mM CuSO4, with approximately 4.96 mg/liter of recombinant VP1 production level. When 2% DMSO or 10 mM sodium butyrate was added at 5 days post-inoculation, the recombinant VP1 production was increased to 8.35 and 5.85 mg/liter, respectively. However, the synergistic effects of DMSO and sodium butyrate were not observed. These results indicate that DMSO and/or sodium butyrate can be successfully used to improve the recombinant HAV VP1 production in culture plates and spinner flasks.

Keyword

Drosophila melanogaster S2 cells; Hepatitis A virus (HAV) VP1; Dimethysulfoxide (DMSO); Sodium butyrate; Spinner flask

MeSH Terms

Butyrates
Capsid Proteins
Dimethyl Sulfoxide
Drosophila
Drosophila melanogaster
Efficiency
Hepatitis
Hepatitis A
Hepatitis A virus
Sodium
Butyrates
Capsid Proteins
Dimethyl Sulfoxide
Sodium

Figure

  • Figure 1 Recombinant VP1 production in DMSO and/or sodium butyrate-supplemented culture of stably transfected S2 cells using culture plates. (A) DMSO (2%) and sodium butyrate (10 mM) were added to the cultures at 4 days post-inoculation. After 2 days incubation, recombinant VP1 expressions in medium fractions were confirmed by Western blot analysis. M: molecular weight marker, Lane 1: the medium fraction of non-treated S2 cells, Lane 2: the medium fraction of 2% DMSO-treated S2 cells, Lane 3: the medium fraction of 10 mM sodium butyrate-treated S2 cells, Lane 4: the medium fraction of 2% DMSO and 10 mM sodium butyrate-treated S2 cells, Lane 5~8: 50, 100, 250, 500 ng of purified recombinant VP1 control from S2 cells. (B) The contents of recombinant VP1 from (A) experiment were estimated and are plotted as a bar diagram. Data are reported as mean ± S.D. Statistically significant differences between treatment and control groups were determined using Student's t test (** p < 0.01).

  • Figure 2 Cell growth in DMSO and/or sodium butyrate-supplemented culture of stably transfected S2 cells using spinner flasks. Data are reported as mean ± S.D. Statistically significant differences between treatment and control groups were determined using Student's t test (* p < 0.05, *** p < 0.001).

  • Figure 3 Recombinant VP1 production in DMSO and/or sodium butyrate-supplemented culture of stably transfected S2 cells using spinner flasks. (A) DMSO (2%) and sodium butyrate (10 mM) were added to the cultures at 5 days post-inoculation. Recombinant VP1 expressions in medium fractions at 9 days post-inoculation were confirmed by Western blot analysis. Lane 1: the medium fraction of non-treated S2 cells, Lane 2: the medium fraction of 2% DMSO-treated S2 cells, Lane 3: the medium fraction of 10 mM sodium butyrate-treated S2 cells, Lane 4: the medium fraction of 2% DMSO and 10 mM sodium butyrate-treated S2 cells, Lane 5-8: 50, 100, 250, 500 ng of purified recombinant VP1 control from S2 cells. (B) The contents of recombinant VP1 from (A) experiment were estimated and are plotted as a bar diagram. Data are reported as mean ± S.D. Statistically significant differences between treatment and control groups were determined using Student's t test (* p < 0.05).


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