title: An in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants.
abstract: Proteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a 'mouse trap' against the host proteases during extraction. After confirming the importance of trypsin- and chymotrypsin-like activities in crude protein extracts of potato (Solanum tuberosum L.) leaves, transgenic lines of potato expressing either tomato cathepsin D inhibitor (CDI) or bovine aprotinin, both active against trypsin and chymotrypsin, were generated by Agrobacterium tumefaciens-mediated genetic transformation. Leaf crude protein extracts from CDI-expressing lines, showing decreased levels of cathepsin D-like and ribulose 1,5-bisphosphate carboxylase/oxygenase hydrolysing activities in vitro, conducted decreased turnover rates of the selection marker protein neomycin phosphotransferase II (NPTII) relative to the turnover rates measured for transgenic lines expressing only the marker protein. A similar stabilizing effect on NPTII was observed in leaf protein extracts from plant lines coexpressing bovine aprotinin, confirming the ability of ectopically expressed broad-spectrum serine proteinase inhibitors to reproduce the protein-stabilizing effect of low-molecular-weight proteinase inhibitors generally added to protein extraction media.
authors.affiliation: CRH/INAF, Département de Phytologie, Pavillon des Services (INAF), Université Laval, Sainte-Foy (Québec), Canada G1K 7P4.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rivard
authors.forename: Raphaël
authors.initials: R
authors.lastname: Anguenot
authors.forename: France
authors.initials: F
authors.lastname: Brunelle
authors.forename: Van Quy
authors.initials: VQ
authors.lastname: Le
authors.forename: Louis-Philippe
authors.initials: LP
authors.lastname: Vézina
authors.forename: Sonia
authors.initials: S
authors.lastname: Trépanier
authors.forename: Dominique
authors.initials: D
authors.lastname: Michaud
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000818:Animals; D007611:Aprotinin / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002417:Cattle; D018551:Solanum lycopersicum / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D010940:Plant Proteins; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D007611:Aprotinin