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Fig. 12.4            Products extracted from tissue/                     primary cellsProduct                   Extracted ...
Fig. 12.5            Problems of extraction from animal/                     human sources     • small quantities availabl...
.17
Lecture 14 - Animal Cell Biotechnology      Animal cell products – Recombinant proteins1. Insulin•   hormone produced by b...
Lecture 14 - Animal Cell Biotechnology            Animal cell products – Recombinant proteinsbeta cells in pancreas      p...
Computer-generated image of insulin hexamers highlighting the threefold symmetry, the zinc ion holdin it together and the ...
Lecture 14 - Animal Cell Biotechnology     Animal cell products – Recombinant proteins• insulin produced from pig pancreas...
Fig. 12.6             Pig to human insulin   A (21)    S- S             S       S              Thr    B (30)   S       S  ...
Producing A and B chains separately
Lecture 14 - Animal Cell Biotechnology   Animal cell products – Recombinant glycoproteins2. Interferons• glycoproteins tha...
Interferon interferes with viral replication in protected cellsButler, M. 1987. Animal cell technology: principles and pro...
Lecture 14 - Animal Cell Biotechnology    Animal cell products – Recombinant glycoproteins3 main types of interferons:1. I...
Lecture 14 - Animal Cell Biotechnology   Animal cell products – Recombinant glycoproteins5. Erythropoietin (EPO)• glycopro...
Physiological role of erythropoietin• Hematopoietic growth factor• Produced in the kidney• Stimulates red blood cell (eryt...
Therapeutic uses of EPOTreatment of anaemia caused by :-•    chronic renal failure•    partial renal failure•    AIDS•    ...
Molecular characteristics of EPO• Molecular weight: 39 kDa• 165 amino acids• Carbohydrate component: 35-40%• 3 N-linked gl...
Fig. 12.11             Structure of erythropoietin
Predicted structure of glycosylated human erythropoietinThe predicted structure of glycosylated protein human Erythropoiet...
Fig. 12.12    Recombinant human Erythropoietin       Non-glycosylated             Glycosylated                            ...
Tetra-antennary N-glycan structure                1-4Asn-X-Ser/Thr                              1-6        1-6   = Fuc    ...
Lecture 14 - Animal Cell Biotechnology    Animal cell products – Recombinant glycoproteins   carbohydrates make up ~40% (...
Fig. 12.13             Variant glycoforms of recombinant Epo and NESP                                                     ...
Fig. 12.15                                                         The biological activity of each isoform of Epo after a ...
Fig. 12.14                                   Serum half-life of analogues of Epo with variable N-glycan sites             ...
Lecture 14 - Animal Cell Biotechnology   Animal cell products – Recombinant glycoproteins3. Plasminogen activators• thromb...
Therapeutic applications• t-PA is used in diseases that feature blood  clots, - - pulmonary embolism  - myocardial infarct...
Fig. 12.9            disulphide bond                   N-glycan
Lecture 14 - Animal Cell Biotechnology   Animal cell products – Recombinant glycoproteins                     Fibrinolysis...
Lecture 14 - Animal Cell Biotechnology   Animal cell products – Recombinant glycoproteins• gene for t-PA transfected into ...
Lecture 14 - Animal Cell Biotechnology   Animal cell products – Recombinant glycoproteins4. Blood-clotting factors• Hemoph...
The clotting cascadeWound surface contactFactor XII       Factor XIIa     Factor XI           Factor XIa             Facto...
Fig. 12.10                         The clotting cascade     Wound surface contactFactor XII               Factor XIIa     ...
Lecture 14 - Animal Cell Biotechnology   Animal cell products – Recombinant glycoproteinsFactor VIII• large glycoprotein (...
Lecture 14 - Animal Cell Biotechnology   Animal cell products – Recombinant glycoproteinsFactor IX• plasma glycoprotein (5...
Lecture 14 - Animal Cell Biotechnology        Animal cell products – Artificial skin• important for skin grafting (i.e. fo...
Fig. 12.16             The principle of gene therapy ex vivo
Lecture 11 recombinant protein production
Lecture 11 recombinant protein production
Lecture 11 recombinant protein production
Lecture 11 recombinant protein production
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Lecture 11 recombinant protein production

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Industrial Microbiology Dr. Butler 2011

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Transcript of "Lecture 11 recombinant protein production"

  1. 1. Fig. 12.4 Products extracted from tissue/ primary cellsProduct Extracted from.... insulin pancreas; bovine or porcine growth hormone human pituitary glands interferon viral activation of cells urokinase human urine factor VIII pooled human blood
  2. 2. Fig. 12.5 Problems of extraction from animal/ human sources • small quantities available • non-human proteins cause immunogenicity • contamination with viruses or prions - Creutzfeld-Jakob disease - HIV from blood
  3. 3. .17
  4. 4. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant proteins1. Insulin• hormone produced by beta cells in the pancreas → allows glucose to pass into cells → suppresses excess production of sugar in the liver and muscles → suppresses breakdown of fat for energy
  5. 5. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant proteinsbeta cells in pancreas preproinsulin proinsulin insulin + C-peptideButler, M. 1987. Animal cell technology: principles and products. Stony Stratford: Open University Press. P107.
  6. 6. Computer-generated image of insulin hexamers highlighting the threefold symmetry, the zinc ion holdin it together and the histidine residues invlolved in zinc- bindingIinsulin51 amino acids5,8808 molecular weight
  7. 7. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant proteins• insulin produced from pig pancreas cells → structure of insulin differs slightly between species → the C-terminal amino acid of the B chain = alanine (threonine in humans)• two problems associated with porcine insulin → causes immunogenic response in some diabetic patients → supply of pancreas fluctuates with meat trade
  8. 8. Fig. 12.6 Pig to human insulin A (21) S- S S S Thr B (30) S S B 30 Ala
  9. 9. Producing A and B chains separately
  10. 10. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteins2. Interferons• glycoproteins that “interfere” with viral propagation in cell cultures• group of small proteins with 140-170 amino acids• secretory protein produced from viral-infected cells, induces antiviral state in neighboring cells
  11. 11. Interferon interferes with viral replication in protected cellsButler, M. 1987. Animal cell technology: principles and products. Stony Stratford: Open University Press. P70.
  12. 12. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteins3 main types of interferons:1. IFN-α (25 subtypes) – produced from β -lymphocytes2. IFN-β – fibroblasts – produced from fibroblasts3. IFN-γ – T-lymphocytes – produced from T-lymphocytes• mode of action not fully understood → synthesis of host enzymes that degrade viral RNA and inhibit protein synthesis
  13. 13. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteins5. Erythropoietin (EPO)• glycoprotein hormone produced by the kidney (hypoxia triggers EPO production)• required for continuous red blood cell production in bone marrow (erythropoiesis)• absence of EPO results in impairment of red blood cell production → anemia• anemia treated with exogenous EPO
  14. 14. Physiological role of erythropoietin• Hematopoietic growth factor• Produced in the kidney• Stimulates red blood cell (erythrocyte) maturation• Induces homodimerization of 2 receptor molecules• Initiates intracellular signalling cascade
  15. 15. Therapeutic uses of EPOTreatment of anaemia caused by :-• chronic renal failure• partial renal failure• AIDS• cancer chemotherapy• autologous transfucion
  16. 16. Molecular characteristics of EPO• Molecular weight: 39 kDa• 165 amino acids• Carbohydrate component: 35-40%• 3 N-linked glycans to Asn at positions 24, 38, 83• 1 O-linked glycan to Ser at position 126
  17. 17. Fig. 12.11 Structure of erythropoietin
  18. 18. Predicted structure of glycosylated human erythropoietinThe predicted structure of glycosylated protein human Erythropoietin . N- and O-glycans wereadded to the core protein structure (pdbid 1BUY) using the Glycoprotein Builder tool at theGLYCAM-Web site (www.glycam.com). High mannose N-linked glycans (Man9GlcNAc2) wereadded at ASN 24, 38 and 83 and one O-linked glycan (a-GalNAc) at Ser126. (R.Woods)
  19. 19. Fig. 12.12 Recombinant human Erythropoietin Non-glycosylated Glycosylated Asn83 Asn38 Asn24 Ser126 39 kDa 18 kDa
  20. 20. Tetra-antennary N-glycan structure 1-4Asn-X-Ser/Thr 1-6 1-6 = Fuc Asn = GlcNAc 1-3 8 = Man 6 1-2 4 = Gal 3 2 = NeuAc 2-3 Linkage position Complex -linkage -linkage tetra-antennary
  21. 21. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteins carbohydrates make up ~40% (by weight) of glycoprotein → important for full activity in vivo allows EPO to remain in circulation (removed by liver) Egrie and Browne (2001) developed a novel form of EPO (novel erythropoiesis-stimulating protein (NESP)) hyper-glycosylated form of EPO with greater half-life (3x half life of EPO)
  22. 22. Fig. 12.13 Variant glycoforms of recombinant Epo and NESP Maximum number of sialic acid groups in glycoform 22 (NESP) 14 12 8 O-linked glycan N-linked glycans
  23. 23. Fig. 12.15 The biological activity of each isoform of Epo after a 30-day treatment Increase in hematocrit from baseline 30 25 20 15 10 5 0 8 9 10 11 12 13 14 Number of sialic acid groups in Epo isoform
  24. 24. Fig. 12.14 Serum half-life of analogues of Epo with variable N-glycan sites 7 6 5 serum half-life (h) 4 3 2 1 0 rEpo 4-glycan NESP Epo type
  25. 25. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteins3. Plasminogen activators• thrombosis (formation of blood clots) is a major cause of premature death• deposition of fibrin in the circulatory system, blocks blood flow• formation of insoluble fibrin controlled by clotting cascade formed during wound healing• t-PA (tissue-plasminogen activator) initiates fibrinolysis (proteolytic cleavage of fibrin)
  26. 26. Therapeutic applications• t-PA is used in diseases that feature blood clots, - - pulmonary embolism - myocardial infarction - stroketo be effective, t-PA must be administered within the first 3 hours/ to be given intravenously,
  27. 27. Fig. 12.9 disulphide bond N-glycan
  28. 28. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteins Fibrinolysis Tissue-plasminogen activator Plasminogen PlasminCoagulation Fibrin Fibrin products (insoluble) (soluble)
  29. 29. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteins• gene for t-PA transfected into CHO-K1 cells, one of the first recombinant products derived from mammalian cells in 1987 → secreted in vivo by a number of tissues → production stimulated by a number of substances, including thrombin and histamine → half-life of t-PA varies from 2-4 min
  30. 30. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteins4. Blood-clotting factors• Hemophilia is a sex-linked (x-chromosome) genetic disease• inactive clotting cascade in blood, can’t form fibrin → hemophilia A – absence of factor VIII → hemophilia B – absence of factor IX
  31. 31. The clotting cascadeWound surface contactFactor XII Factor XIIa Factor XI Factor XIa Factor IX Factor IXa +Factor VIII + Thrombin Factor X Factor Xa +Factor V Prothrombin Thrombin Fibrinogen Fibrin clot
  32. 32. Fig. 12.10 The clotting cascade Wound surface contactFactor XII Factor XIIa Factor XI Factor XIa Factor IX Factor IXa + Factor VIII + Thrombin Factor X Factor Xa +Factor V Prothrombin Thrombin Fibrinogen Fibrin clot
  33. 33. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteinsFactor VIII• large glycoprotein (265 kDa)• gene – 186 kB, 26 exons, 25 introns (overlapping strands of DNA from genomic and cDNA aligned, without introns)• BHK cells transfected with expression vector containing gene encoding Factor VIII• produces biologically active protein with correct tertiary folding and glycosylation• stabilized by addition of Willebrand factor, normally found as a combined protein complex in blood
  34. 34. Lecture 14 - Animal Cell Biotechnology Animal cell products – Recombinant glycoproteinsFactor IX• plasma glycoprotein (57 kDa) secreted by hepatocytes• called “Christmas factor”, after first family diagnosed with clotting deficiency• gene cloned into rat hepatoma cell line → contains enzymes for post-translation modifications
  35. 35. Lecture 14 - Animal Cell Biotechnology Animal cell products – Artificial skin• important for skin grafting (i.e. for severe burn victims)• one method described by Hardin-Young and Parenteau 2002) → dermal-equivalent formed from fibroblasts → epidermal equivalent formed from keratinocytes• keratinocytes and fibroblasts are derived from neonatal foreskin tissue, lack antigen presentation
  36. 36. Fig. 12.16 The principle of gene therapy ex vivo
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