title: Expression of recombinant cellulase Cel5A from Trichoderma reesei in tobacco plants.
abstract: Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.
authors: Institute for Molecular Biotechnology, Bio7, RWTH Aachen University.
authors: Megan
authors: M
authors: Garvey
authors: Institute for Molecular Biotechnology, Bio7, RWTH Aachen University.
authors: Johannes
authors: J
authors: Klinger
authors: Institute for Molecular Biotechnology, Bio7, RWTH Aachen University.
authors: Holger
authors: H
authors: Klose
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology.
authors: Rainer
authors: R
authors: Fischer
authors: Institute for Molecular Biotechnology, Bio7, RWTH Aachen University; commandeur.ulrich@molbiotech.rwth-aachen.de.
authors: Ulrich
authors: U
authors: Commandeur
mesh_terms: D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D002480:Cellulase