4. production of recomb. proteins


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4. production of recomb. proteins

  1. 1. Production of Recombinant Therapeutic Proteins
  2. 2. O utline1 . Introduction2. Methods used to produce recombinant proteins3. H ow are transgenic animals produced?4. Advantage of transgenic systems5. L im itations of the transgenic expression system s6. List of therapeutic proteins
  3. 3. 1.INTRODUCTIONProduction of Recombinant Therapeutic ProteinsRecombinant DNA technology is widely used in theproduction of therapeutic agents such as;hormones, cytokines, growth factors, antibiotics,vaccines, blood products like albumin, thrombolytics,fibrynolytics, clotting factors such as factor VII, factorIX, tissue plasminogen activator and many more.
  4. 4. • All these therapeutic agents can be produced in a large quantity and also more economically by using rDNA technology.• Many proteins which may be used for medical treatment or for research are normally expressed at very low concentrations.• Through rDNA technology, a large quantity of proteins can be produced. This involves inserting the desired protein gene into an "expression vector" which must contain a promoter so that the protein can be expressed.
  5. 5. 2. METHODS USED TO PRODUCE RECOMBINANT PROTEINS (i) Production of recombinant proteins in microbial bioreactorsExamples E.coli expression system Saccharomyces cerevisiae(ii) Mammalian cell derived bioreactors E.g. Chinese Hamster Ovary cell (CHO) bioreactors. (iii) Animal Bioreactors “Pharming”Production of Recombinant Therapeutic Proteins in the Milk of Transgenic Animals Eg, Cows, sheep, pigs etc.
  6. 6. (i) Microbial bioreactors• The first microbial bioreactors, in particular Escherichia coli (bacterial) and Saccharomyces cerevisiae & Pichia pastoris (yeasts) were found to be satisfactory for the production of simple polypeptides such as insulin and human growth hormone• However, microbial bioreactors were found to be unsuitable for proteins with complex post- translational modifications or intricate folding requirements, such as the coagulation factors, or monoclonal antibodies.
  7. 7. • This led to the development of large-scale mammalian cell culture, for example, the use of Chinese Hamster Ovary (CHO) cell cuture bioreactors.Limitations of microbial bioreactors• Bacteria often improperly fold complex proteins, leading to involved and expensive refolding processes and ;• Both bacteria and yeast lack adequate post- translational modification machinery for mammalian- specific N- and O-linked glycosylation, γ- carboxylation, and proteolytic processing
  8. 8. Building the Transgenes ON/OFF Switch Makes Protein stop signPROMOTER INTRON CODING SEQUENCE poly A signal Transgene Selectable Marker Gene Plasmid DNA Constructbacterial genes •antibiotic marker •replication origin
  9. 9. An overview of the recombination process in Escherichia coli bioreactor
  10. 10. (ii) Cell culture bioreactors• These technologies permitted the development of numerous monoclonal antibodies, cytokines, and other complex bioactive biomolecules.• However, there are proteins that, due to a combination of complex structure and large therapeutic dosing have until now eluded (fail to be attained) recombinant production using traditional bacterial and cell culture bioreactors
  11. 11. • E.g Commercial recombinant production of complex molecules, such as antithrombin and alpha1-antitrypsin, has not yet been achieved in microbial or mammalian cell derived bioreactors• Cell culture systems require high initial capital expenditures, lack scale-up (or down) flexibility and use large volumes of culture media• This led to development of transgenics technology i.e Production of Recombinant Therapeutic Proteins in the Milk of Transgenic Animals
  12. 12. (iii) Production of Recombinant Therapeutic Proteins in the Milk of Transgenic AnimalsWhat is a transgenic animal?• A transgenic animal is one which has been genetically altered to have specific characteristics (genes) it otherwise would not have.• Different types of transgenic animals have been invented to carter to specific societal needs.• It includes; transgenic disease models, transgenic pharmers, xenotransplanters and transgenic food
  13. 13. 3. How are transgenic animals produced?The foreign DNA can be inserted in three ways:(iii)DNA microinjectionFusing an expression vector, comprising a gene that is encoded for the human or humanized target protein with mammary gland-specific regulatory sequences, and then inserting into the germline of the selected production species.
  14. 14. • When integrated, the milk-specific expression construct becomes a dominant genetic characteristic that is inherited by the progeny of the founder animal (Figure 1).• This general strategy makes it possible to harness the ability of dairy animal mammary glands to produce large quantities of complex proteins.
  15. 15. Fig 1.
  16. 16. Electrofusion: Fusion induced by electric pulse fusion pulse fusion productCells brought close Heterokaryon phase:together nuclei distinct
  17. 17. Cont………(ii) Retrovirus-Mediated Gene Transfer• A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA• retroviruses used as vectors to transfer genetic material into the host cell, resulting in a chimera• chimeras are inbred for as many as 20 generations until homozygous transgenic offspring are born
  18. 18. (iii) Embryonic Stem Cell-Mediated Gene Transfer• This method involves isolation of totipotent stem cells from embryos• The desired gene is inserted into these cells• Cells containing the desired DNA are incorporated into the host’s embryo, resulting in a chimeric animal
  19. 19. Advantage of transgenic systems• Transgenic livestock can be maintained and scaled- up in relatively inexpensive facilities• Use animal feed as raw material• Can achieve impressive yields of recombinant proteins.
  20. 20. Limitations of the transgenic expression systems• Limitations are related to potential adverse effects of bioactive heterologous proteins on the health of the production animals and to a lesser extent, to initial timelines. E.g. 12-18 months in goats• Although transgenic expression systems are able to perform complex post-translational modifications, such as γ-carboxylation, β-hydroxylation or N- and O-linked glycosylation,
  21. 21. -there are species-specific and tissue-specificcharacteristics for these modifications that may affectthe appropriateness of a given system for theexpression of specific proteins.This is also a challenge found with mammalian cellculture, microbial expression systems or transgenicplants.
  22. 22. •Transgenic goatsProducing anti-thrombin(rhAT) therapeutic protein.• It is used to treat clottingdefects as in Haemophiliaand is used to preventDIC and DVT also usedbefore surgery
  23. 23. Webster and Peter Transgenic youngNexia Biotechnologies transferred male goats carryingthe silk gene from Orb spiders into silk genegoatsThe resulting male goats were usedto sire silk-producing female goatsEach goat produces several grams ofsilk protein in her milkThe silk is extracted, dried to a whitepowder and spun into fibersThe fibers are stronger and moreflexible than steel
  24. 24. Tracy the sheep (1997).The first transgenic animal to produce a recombinantprotein drug in her milk alpha-1-antitrypsin (AAT)treatment for emphysema & cystic fibrosis. Created byPPL Therapeutics & The Roslin Institute
  25. 25. Herman the bull-15/12/1990: first transgenic bullcarrying transgene for humanlactoferrin (Gene Pharming, TheNetherlands)-Dec 1992: permission togenerate 50 offsprings by AI-Oct 1993: first calf born-March 1996: 5 cows producinghuman lactoferrin in their milk
  26. 26. •Some Biotherapeutic companies has received approval to sellhuman anti-thrombin (hAT) purified from goat’s milk in Europe•Technology is not restricted to cows,goats, & sheep, there is interest inusing rabbits since housing costs aresignificantly less & generation time is faster•Chickens which produce recombinant drugs in their eggshave been produced by The Roslin Institute.
  27. 27. = Is it good to make transgenics from Mr. D.O.G?? THE END