Production of a sugarcane cystatin in Nicotiana benthamiana

• Grant number: 18/20051-2
• Support type: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): March 11, 2019
• Effective date (End): September 10, 2019
• Field of knowledge: Biological Sciences - Biology
• Principal researcher: Flavio Henrique da Silva
• Grantee: Priscila Yumi Tanaka Shibao
• Supervisor abroad: Andreas Schiermeyer
• Home Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Research place: Fraunhofer-Gesellschaft, Germany
• Associated to the scholarship: 17/16118-1 - Production of Canecystatin in transgenic sugarcane vacuoles, BP.DR

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.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

TANAKA SHIBAO, PRISCILA YUMI; SANTOS-JUNIOR, CELIO DIAS; SANTIAGO, ADELITA CAROLINA; MOHAN, CHAKRAVARTHI; MIGUEL, MARIANA CARDOSO; TOYAMA, DANYELLE; SANCHES VIEIRA, MARCOS ANTONIO; NARAYANAN, SUBRAMONIAN; FIGUEIRA, ANTONIO; CARMONA, ADRIANA K.; et al. Sugarcane cystatins: From discovery to biotechnological applications. International Journal of Biological Macromolecules, v. 167, p. 676-686, JAN 15 2021. (17/16118-1, 19/11683-8, 15/10855-9, 18/20051-2)