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Recombinant protein expression seminar paper

a seminar presentation paper

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Recombinant protein expression seminar paper

  2. 2. INTRODUCTION• RPE is a subcomponent of gene expression which involves translation of the gene into polypeptide chains and folding into proteins• Used in esp. enzyme production, insulin, endonucleases, DNA pol, reverse T.• Classified according to expression systems; bacterial, yeast, insect, mammalian.
  4. 4. Bacterial expression• Isolation of total or poly A tail RNA from cells or tissue• First cDNA
  5. 5. Bacterial expression cont’• Addition of oligonucleotide plugs
  6. 6. Bacterial expression cont’• PCR amplification of cDNA
  7. 7. Bacterial expression cont’• Directional ligation into appropriate vector Periplasmic export extracellular export
  8. 8. Bacterial expression cont’• Amplification of the primary library into a bacteria of choice, E. coli, B. subtilis.• Transformation done through heat shock, electroporation. Heat shock transformation
  9. 9. Bacterial expression cont’• Protein expression recovery sites in E. coli- cytoplasmic, periplasmic, medium
  10. 10. Bacteria expression cont’• Harvesting done by• Chemical• Enzymatic e.g lysozymes• Biochemical e.g osmotic shock• Physical e.g. sonication• Mutant E. coli strains, L-forms
  11. 11. Bacterial expression cont’• Advantages and disadvantages in the protein production into each compartment.
  12. 12. Bacterial expression cont’• Purification-affinity chromatography• Quality control:• Acrylamide gel,• SDS-PAGE,• Western blot. 2 1 3 4
  13. 13. Yeast expression• Same protocol as bacteria but the host is yeast cells where your protein is harvested
  14. 14. Expression in yeast cont’• Genes synthesis• cDNA cloning in Yeast expression factor and tagging• Transformation in yeast cells• Confirmation of pos. trans-formants by PCR• Affinity purification• SDS-PAGE/ western blot
  15. 15. Insect expression
  16. 16. Insect expression cont’• Gene synthesis• cDNA cloning in baculovirus expression vector• Recombinant virus generation and recombinant protein in bacmid• Transfection of insects cells with recombinant bacmid• Generation of virus stock in insect cells• Quality control, SDS-PAGE/ western blot
  17. 17. Mammalian expression
  18. 18. Mammalian expression• Gene synthesis• cDNA cloning into an expression factor• Transfection of cells with expression vector• Evaluation of expression by western blotting• Bio-reactor production• Harvesting• Affinity chromatography• Quality control, SDS-PAGE/ western blot
  19. 19. Expression in plants
  20. 20. Conclusion
  21. 21. References• Artes Biotechnology Langenfeld, Germany, http://www.artes-• Dual SystemsBiotech, Schleren, Switzerland. for-escherichia-coli.html• Genscript, the biology GRO, Piscataway, USA.• Joan L. &J. Foster (2009) Microbiology, an evolving science. W.W. Norton, New York, USA 2nd ed.• Park, S. J. and Lee, S. Y., "Efficient recovery of secretory recombinant protein from protease negative mutant Eshcherichia coli" Biotechnol. Techniques, 64(12):4897- 4903 (1998)• Tokyo Future Style Inc.
  22. 22. Merry-xmas