Production of a sugarcane cystatin in Nicotiana benthamiana

Abstract

Plant cystatins (PhysCys) are tight-binding cysteine protease inhibitors produced by plants. Besides presenting cystatins conserved motifs, they also show an active L-A-R-F-A-V-X (3)-N-terminal sequence that is folded in an alpha-helix. Up to date, Laboratory of Molecular Biology has discovered, produced and characterized six sugarcane cystatins named CaneCPI-1 to CaneCPI-6. Among them, CaneCPI-5 presents prominent usage in dental enamel protection against erosion and is currently under patent licensing. However, the obtained yield from Escherichia coli and Pichia pastoris is still poor for industrial supply. In this sense, molecular farming is the most suitable option for scaling-up its production yield. The transient transformation of Nicotiana benthamiana offers rapid production of recombinant proteins through the infiltration of engineered agrobacteria into leaf tissue. The target proteins can be extracted after an incubation period of a few days and extracted and recovered from the infiltrated tissue. Therefore, the present study aims to produce CaneCPI-5 in Nicotiana benthamiana plants by transient transformation and in parallel by stable transformation of tobacco BY-2 suspension cells as a second option. After confirming protein production by immunoblot, the protein will be purified using nickel affinity columns and CaneCPI-5 activity will be assessed using fluorimetric assays measuring substrate Z-Phe-Arg-AMC releasing in presence of the model cysteine protease papain. In summary, this project employs state of the art molecular farming methods to obtain a protein of high commercial value.