Expression of recombinant cellulase Cel5A from Trichoderma reesei in tobacco plants

J Vis Exp. 2014 Jun 13:(88):51711. doi: 10.3791/51711.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Cellulase / biosynthesis*
  • Cellulase / genetics
  • Nicotiana / enzymology*
  • Nicotiana / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Trichoderma / enzymology*
  • Trichoderma / genetics

Substances

  • Recombinant Proteins
  • Cellulase