85FC4CA3d01 - 5.9 Production of recombinant proteins in...

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5.9. Production of recombinant proteins in milk and transgenesis and the environment - Important in treatment of certain congenital disorders haemophylia A (F-VIII) emphysema ( α 1 –AT) Pompe disease ( α -glucosidase) - complex molecules ( glycosylated /other modifications) - Advantages milk over serum - less complex - expression with high efficiëncy - specific expression by choice correct promoter - post-translational modifications in epithelial cells
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- mice : e.g. rabbit - WAP promoter + h-GH TG 8 mg/ml + α 1-AT TG finally grams! Lactating animals! Milking problem! - Rabbits : larger volumes possible : liters/ rabbit/ year 8 lactations/year each 4W e.g. h-IGF1: grams! α glucosidase (5.000-10.000 patiënts) 500-1000 rabbits for all patiënts
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- goats : eg La-tPA after β casein promoter 4,5 mg/ml at 1000 ml/day
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Milk : in general: milk consists of - salts eg 2 mg/ml Ca ++ - 40 mg/ml lactose - 50 mg/ml proteins caseïns (micells) α , β lactoglobulins immunoglobulins plasma proteins (albumin) proteases purification - remove fat by centrifugation - remove cells ( 10 5 -10 6 /ml) by centrifugation - precipitation of caseïns via pH lowering filtration, etc.
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recent examples : 1) lactose - 70% of population has problem metabolizing lactose (abdominal pain,...) - remove lactose (ultracentrifugation, precipitation, degradation β -gal) - alternatively: transgenic overexpression of lactose-phlorzine-hydrolase (lactase activity) gene after α -lactalbumine promotor: 50-85 % reduction lactose.
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2) 2006: ‘European Medicines Agency’ first time approval for use of a product from milk of Tg animal in clinic ‘Atryn’ = human antitrombin from milk of Tg goats in treatment of antithrombin-deficient patients
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Transgenesis and the environment - Phosphates in excretions of pigs > eutrophication, hypoxia... - Let animals take up more phosphates from food by degradation of phytate via phytase - Add recombinant phytase to food: very expensive, unstable Phytase ( E.coli ) TG mouse via const. or inducible promoter - Expression in salivary glands - Glycosylated - % phosphate from 2.31 ± 0.07 (wt) to 2.06 ± 0.06 Now also in Tg pigs ( Enviropig )
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5.10. Study of promotor activity via reporter genes deletions in construct generate Tg mice and study expression truncated promoters pointmutations in promoters promoter Reporter gene
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reporter genes : CAT in cell lysates acetylation of C 14 - chloramphenicol also on tissue sections LAC Z β galactosidase gene of E. coli simple detection/especially in situ /developmental biology adult animals: expression blocked somewhat background only fixed tissues/slides/not living Mhox (mesenchym of developing limbs + cranofacial region)
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luciferase several versions used a lot genes from fire flies, mushrooms, fish, … emission of photons from luciferin (+ATP) in cell lysates /sections also at level of complete organism (e.g. CCD camera)
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hPLAP heat stabile no repression in mouse cells membrane bound (neurons!)
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GFP - more and more - early problems (thermo-instability, low fluorescence, poor solubility) - later mutant versions: EGFP, … stable toxicity problems (eg heart problems after overexpression after α MHC promoter), by H 2 O 2 ??
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