Transcript of "Lecture 11 recombinant protein production"
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
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
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
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.
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
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
Fig. 12.6 Pig to human insulin A (21) S- S S S Thr B (30) S S B 30 Ala
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
Interferon interferes with viral replication in protected cellsButler, M. 1987. Animal cell technology: principles and products. Stony Stratford: Open University Press. P70.
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
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
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
Therapeutic uses of EPOTreatment of anaemia caused by :-• chronic renal failure• partial renal failure• AIDS• cancer chemotherapy• autologous transfucion
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
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)
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)
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
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
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
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)
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,
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
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
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
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
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
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
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
Fig. 12.16 The principle of gene therapy ex vivo
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