les6 - 5.9. Production of recombinant proteins in milk and...

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5.9. Production of recombinant proteins in milk and transgenesis and the environment - Important in treatment of certain congenital disorders hemophylia A (F-IX) emphysema ( α 1 –AT) Pompe disease ( α -glucosidase) - complex molecules ( glycosyl. /other modifications) - Advantages milk over serum - less complex - expression with high efficiëncy - specific expression by choice correct promotor - post-translational modifications in epithelial cells
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mice : e.g. rabbit - WAP promotor + h-GH TG 8mg/ml + α 1-AT TG finally grams! Lactating animals! Melking problem! Rabbits : larger volumes possible : liters/ rabbit/ year 8 lactations/year each 4W eg 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 promotor 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 cellc ( 10 5 -10 6 /ml) by centrifugation - precipitation of caseïns via pH lowering filtration, etc.
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recent example : milk contains lactose - 70% of population has problem metabolizing lactose (abdominal pain,. ..) - remove lactose (ultracentr., precipitation, degradation β -gal) - alternatively: transgenic overexpr. of lactose-phlorzine- hydrolase (lactase activity) gene after α -lactalbumine promotor: 50-85 % reduction lactose.
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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 environment - Phosphates in excretions of pigs > eutrophiation, hypoxia. .. - Let animals take up more phosphates from food by degradation phytate via phytase - Add recombinant phytase to food: very expensive, unstable Phytase ( E.coli ) TG mouse via const. or ind. promotor - Expression in salivary glands - Glycosylated - % phosphate from 2.31 ± 0.07 (wt) to 2.06 ± 0.06
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5.10. Study of promotor activity via reporter genes deletions in construct generate Tg mice and study expression truncated prom. pointmutations promotor Reporter gene
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reporter genes : CAT in cell lysates acetyl. of C14 chloramphenicol also on tissue sections LAC Z β galactosidase gene of E. coli simple detection/esp. in situ /developm. biol. adult animals: expression blocked somewhat background only fixed tissues/slides/not living Mhox (mesenchym of developing limbs + cranofacial region)
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luciferase used a lot gene from fire flies emission of photons from luciferrin (+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-instrability, low fluorescence, poor solubility) later mutant versions: EGFP, … stable toxicity problems (eg heart problems after overexpression after
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les6 - 5.9. Production of recombinant proteins in milk and...

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