Summary
Recombinant plasmids containing a complementary deoxyribonucleic acid coding mouse endostatin were transfected and stably expressed in Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing recombinant endostatin were isolated after 4 wk of selection with hygromycin B. Recombinant endostatin expressed in the stably transformed S2 cells under the influence of the Drosophila BiP protein signal sequence was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at maximum inhibition for recombinant endostatin was approximately 1.8 μg/ml. The stably transformed S2 cells produced 18 mg recombinant endostatin/L 7 d after induction with 5 μM CdCl2. Sodium butyrate supplementation (2.5 mM) increased recombinant endostatin production by 17%. These findings demonstrate optimal production and in vitro activity of recombinant endostatin from stably transformation D. melanogaster S2 cells.
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Arnold, F. H. Metal-affinity separations: a new dimension in protein processing. Biotechnology 9: 151–156; 1991.
Arts, J.; Lansink, M.; Grimbergen, J.; Toet, K. H.; Kooistra, T. Stimulation of tissue plasminogen activator gene expression by sodium butyrate and trichostatin A in human endothelial cells involves histone acetylation. Biochem. J. 310: 171–176; 1995.
Blezinger, P.; Wang, J.; Gondo, M., et al. Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene. Nat. Biotechnol. 17: 343–348; 1999.
Boehm, T.; Folkman, J.; Browder, T.; O'Reilly, M. S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390: 404–407; 1997.
Boehm, T.; O'Reilly, M. S.; Keough, K.; Shiloach, J.; Shapiro, R.; Folkman, J. Zinc-binding of endostatin is essential for its antiangiogenic activity. Biochem. Biophys. Res. Commun. 252: 190–194; 1998.
Boehm, T.; Pirie-Shepherd, S.; Trinh, L. B.; Shiloach, J.; Folkman, J. Disruption of the KEX1 gene in Pichia pastoris allows expression of full-length murine and human endostatin. Yeast 15: 563–567; 1999.
Canaan-Haden, L.; Ayala, M.; Fernandez-de-Cossio, M. E.; Pedroso, I.; Rodes, L.; Gavilondo, J. V. Purification and application of a single-chain Fv antibody fragment specific hepatitis B virus surface antigen. Bio Techniques 19: 606–614; 1995.
Diaz-Flores, L.; Gutierrez, R.; Varela, H. Angiogenesis: an update. Histol. Histopath. 9: 807–843; 1994.
Dorner, A. J.; Wasley, L. C.; Kaufman, R. J. Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyrate-treated Chinese hamster ovary cells. J. Biol. Chem. 264: 20,602–20,607; 1989.
Kerbel, R. S. A cancer therapy resistant to resistance. Nature 390: 335–336; 1997.
Laemmli, U. K. Cleavage of structural proteins during the assembly of head of bacteriophage. Nature 227: 680–685; 1970.
Oh, S. K. W.; Vig, P.; Chua, F.; Teo, W. K.; Yap, M. G. S. Substantial over-production of antibodies by applying osmotic pressure and sodium butyrate. Biotechnol. Bioeng. 42: 601–610; 1993.
O'Reilly, M. S.; Boehm, T.; Shing, Y., et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88: 277–285; 1977.
Park, J. H.; Kim, H. Y.; Han, K. H.; Chung, I. S. Optimization of transfection conditions for expression of green fluorescent protein in Drosophila melanogaster S2 cells. Enzyme Microb. Technol. 25: 558–563; 1999.
Park, J. H.; Lee, J. M.; Chung, I. S. Production of recombinant endostatin from stably transformed Drosophila melanogaster S2 cells. Biotechnol. Lett. 21: 729–733; 1999.
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Park, J.H., Chang, K.H., Lee, J.M. et al. Optimal production and in vitro activity of recombinat endostatin from stably transformed Drosophila melanogaster S2 cells. In Vitro Cell.Dev.Biol.-Animal 37, 5–9 (2001). https://doi.org/10.1290/1071-2690(2001)037<0005:OPAIVA>2.0.CO;2
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DOI: https://doi.org/10.1290/1071-2690(2001)037<0005:OPAIVA>2.0.CO;2