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Molecular Pharming: High Level Plant Protein Expression (CPMV-HT) »
Enzymes: Gastric Lipase
Enzymes: Gastric Lipase
Expression of enzymes
A number of active enzymes have been successfully produced in plants using the pEAQ system. Here are a few examples:
Human Gastric Lipase
Human gastric lipase (hGL) is a member of the family of preduodenal lipases, primarily responsible for initiation of lipolysis of dietary fats, such as triglycerides in the gastrointestinal tract. It is a candidate for treatment of pancreatic enzyme insufficiency, as a superior substitute for the current treatment that uses enzyme obtained from porcine pancreatic extract. Recently, it was demonstrated that active recombinant hGL can be produced in N. benthamiana by transient expression using the CPMV-HT system.
Enzyme assays confirmed that the plant-produced hGL was active. The figure below shows results of an assay conducted to detect the presence of lipase activity in clarified leaf extracts (performed at pH 4.0 using MU-oleate as a substrate). It was seen that extracts from leaves infiltrated with either the wt-hGL or hGL-His pEAQ-HT-based constructs had the ability to release the MU moiety from the MU-oleate substrate, demonstrating that the plant-produced lipase was active.
Reference: Vardakou, M., Sainsbury, F., Rigby, N., Mulholland, F. and Lomonossoff, G. P. (2012). Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system. Protein Expression and Purification 81: 69-74.
Avena sativa oxidosqualene cyclase (OSC)
Oxidosqualene cyclase and CYP450 from oat have been successfully expressed in N. benthamiana leaves. Expression of oxidosqualene cyclase alone resulted in production of β-amyrin, a metabolite not normally produced by this species. This shows the ability of pEAQ vectors to express metabolic enzymes for understanding their function and to produce novel compounds.
Reference: Sainsbury F, Saxena P, Geisler K, Osbourn A and Lomonossoff GP (2012). Using a virus-derived system to manipulate plant natural product biosynthetic pathways. Methods in Enzymology; 517: 185-202.
Rice chitinase OsChia4a
Isolated enzyme shown to have anti-fungal activity
Reference: Miyamoto K, Shimizu T, Lin F, Sainsbury F, Thuenemann E, Lomonossoff G, Nojiri H, Yamane H, and Okada K (2012). Identification of an E-box motif responsible for the expression of jasmonic acid-induced chitinase gene OsChia4a in rice. Journal of Plant Physiology; 169: 621-627.
Artemisia annua amorpha-3,11-diene synthase (ADS) and epi-cedrol synthase (ECS)
Enzymes for the conversion of farnesyl diphosphate into amorpha-4,11-diene and epi-cedrol, respectively have been produced in plants using the pEAQ system.
Reference: Kanagarajan S, Muthusamy S, Gliszczynska A, Lundgren A, and Brodelius PE (2012). Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana. Plant Cell Rep; 31(7): 1309-1319.