Biotechnology and Bioprocess Engineering:BBE

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
권호 표지

Expression of a Functional Human Tumor Necrosis Factor-${\alpha}$ (hTNF-$\alpha$) in Yeast Saccharomyces cerevisiae

  • Park, Seung-Moon (Institute for Molecular Biology and Genetics, Basic Science Research Institute) ;
  • Mo, Ae-Young (Institute for Molecular Biology and Genetics, Basic Science Research Institut) ;
  • Jang, Yong-Suk (Bank for Cytokine Research, Chonbuk National University) ;
  • Lee, Jae-Hwa (Department of Biotechnology, College of Engineering, Silla University) ;
  • Yang, Moon-Sik (Institute for Molecular Biology and Genetics, Basic Science Research Institute) ;
  • Kim, Dae-Hyuk (Institute for Molecular Biology and Genetics, Basic Science Research Institute)
  • Published : 2004.07.01
Download PDF
⟨ Previous Next ⟩

Abstract

The recombinant soluble human tumor necrosis factor-alpha (hTNF-$\alpha$) was expressed in a yeast Saccharomyces cerevisiae and its cytotoxicity was evaluated. A cDNA encoding hTNF-$\alpha$ was placed under the control of two different promoters: a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD promoter, consisting of alcohol dehydrogenase II (ADH2) and the GPD promoter. A Northern blot analysis revealed that, although variation in the expression level of hTNF-$\alpha$ existed among transformants, the higher expression was obtained with the GPD promoter. Expressed hTNF-$\alpha$ protein (rhTNF-$\alpha$) was successfully secreted into the culture medium, producing 2.5 mg per liter of culture filtrate, with no changes in cell growth. The bioassay for observing the cytotoxicity to the murine L929 fibroblast cell line, with serial dilution of rhTNF-$\alpha$, indicated that the secreted rhTNF-$\alpha$ was bioactive and its dose-response was improved eight to ten times over that of the E. coli-derived rhTNF-$\alpha$.

Keywords

References

  1. Annu. Rev. Med. v.45 Tumor necrosis factor: A pleiotropic cytokine and therapeutic target Tracey, K. J.;A. Cerami https://doi.org/10.1146/annurev.med.45.1.491
  2. Trends Biotechnol. v.9 The prospects for therapy with tumor necrosis factors and their antagonists Porter, A. G. https://doi.org/10.1016/0167-7799(91)90053-K
  3. J. Biol. Chem. v.259 Human lymphotoxin. Production by a lymphoblastoid cell line, purification, and initial characterization Aggarwal, B. B.;B. Moffat;R. N. Harkins
  4. J. Immunol. v.135 Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo Beutler, A. B.;W. I. Milsark;A. Cerami
  5. Eur. J. Cancer Clin. Oncol. v.25 Phase I study of intratumoral application of recombinant human tumor necrosis factor Pfreundschuh, M. G.;H. T. Steinmetz;R. Tuschen;V. Schenk;V. Diehl;M. Schaadt https://doi.org/10.1016/0277-5379(89)90034-5
  6. Proc. Natl. Acad. Sci. USA v.81 Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae Brake, A. J.;J. P. Merryweather;D. G. Coit;U. A. Heberlein;T. P. Masiary;G. T. Mullenback;M. S. Urdea;P. Valenzuela;P. J. Barr https://doi.org/10.1073/pnas.81.15.4642
  7. Biotechnol. Bioprocess Eng. v.7 Solid-phase refolding of poly-lysine tagged fusion protein of hEGF and angiogenin Park, S. J.;K. Ryu;C. W. Suh;Y. G. Chai;O. B. Kwon;S. K. Park;E. K. Lee https://doi.org/10.1007/BF02935871
  8. Biotechnol. Bioprocess Eng. v.8 Secretory production of hGM-CSF with a high specific biological activity by transgenic plant cell suspension culture Kwon, T. H.;Y. M. Shin;Y. S. Kim;Y. S. Jang;M. S. Yang https://doi.org/10.1007/BF02940269
  9. J. Microbiol. Biotechnol. v.10 Expression of Murine GM-CSF from Recombinant Aspergillus niger Kim, M. J.;T. H. Kwon;Y. S. Jang;M. S. Yang;D. H. Kim
  10. Molecular Cloning Sambrook, J.;E. F. Fritsch;T. Maniatis
  11. Enzyme Microb. Technol. v.30 Expression of a functional human interleukin-18 in yeast Lim, Y. Y.;M. Y. Lee;B. W. Chung;S. M. Park;S. G. Park;Y. S. Jang;M. S. Yang;D. H. Kim https://doi.org/10.1016/S0141-0229(02)00043-1
  12. J. Biotechnol. v.81 Expression of glucose oxidase by using recombinant yeast Park, E. H.;Y. M. Shin;Y. Y. Lim;T. H. Kwon;D. H. Kim;M. S. Yang https://doi.org/10.1016/S0168-1656(00)00266-2
  13. Gene v.94 Expression and secretion of rice-alpha-amylase by Sacchromyces cerevisiae Kumagai, M. H.;M. Shah;M. Terashima;Z. Vrkljan;J. R. Whitaker;R. L. Rodriguez https://doi.org/10.1016/0378-1119(90)90389-9
  14. J. Microbiol. Biotechnol. v.11 Enhanced and targeted expression of fungal phytase in Sacchromyces cerevisiae Lim,Y.Y.;E.H.Park;J.H.Kim;S.M.Park;H.S.Jang;Y.J.Park;S.W.Yoon;M.S.Yang;D.H.Kim
  15. Biotechniques v.22 Simultaneous introduction of multiple mutations using overlap extension PCR Ge, L.;P. Rudolph
  16. J. Bacteriol. v.153 Transformation of intact yeast cells treated with alkali cations Ito, H.;Y. Fukuda;K. Murata;A. Kimura
  17. Proc. Natl. Acad. Sci. USA v.81 Genomic sequencing Church, G. M.;W. Gilbert https://doi.org/10.1073/pnas.81.7.1991
  18. Anal. Biochem. v.132 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity Feinberg, A. P.;B. Vogelstein https://doi.org/10.1016/0003-2697(83)90418-9
  19. Methods Enzymol. v.182 Concentration of proteins and removal of solutes Pohl, T. https://doi.org/10.1016/0076-6879(90)82009-Q
  20. Anal. Biochem. v.72 A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding Bradford, M. M. https://doi.org/10.1016/0003-2697(76)90527-3
  21. J. Immunol. Methods v.65 Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays Mosmann, T. https://doi.org/10.1016/0022-1759(83)90303-4
  22. Yeast v.5 High level of expression of a protective antigen of schistosomes in Saccharomyces cerevisiae Loison, G.;A. Vidal;A. Findeli;C. Roitsch;J. M. Balloul;Y. Lemoine https://doi.org/10.1002/yea.320050609
  23. Nucleic Acids Res. v.15 Translation and stability of an Escherichia coli beta-galactosidase mRNA expressed under the control of pyruvate kinase sequences in Saccharomyces cerevisiae Purvis, I. J.;A. J. E. Bettany;L. Loughlin;A. J. P. Brown https://doi.org/10.1093/nar/15.19.7963
  24. Immunother. Realized v.65 Lymphokines and cytokines as cancer treatment Borden, E. C.;P. M. Sondel
  25. Natl. Immun. Cell Growth Regul. v.9 Lessons from the clinical trails of interleuking-2 Parkinson, D. R.
  26. J. Am. Med. Assoc. v.271 Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin 2 Rosenberg, S. A.;J. C. Yang;S. L. Topalian;D. J. Schwartzentruber;J. S. Weber;D. R. Parkinson;C. S. Seipp;J. H. Einhorn;D. E. White https://doi.org/10.1001/jama.271.12.907