Transgenic Nicotiana tabacum seeds expressing the Mycobacterium tuberculosis Alanine- and Proline-rich antigen

Diego G Módolo,José A Yunes,Sylvia M De Sousa,Cynthia S Horn,José S M Soares,Marcelo Menossi,Leila M Lima

doi:10.1186/s13568-018-0708-y

Abstract

The glycoprotein APA (Alanine- and Proline-rich Antigen, a 45/47 kDa antigen complex, Rv1860) is considered as a major immunodominant antigen secreted by M. tuberculosis. This antigen has proved to be highly immunogenic in experimental models and humans, presenting a significant potential for further development of a new vaccine for tuberculosis. Glycosylation plays a key role in the immunogenicity of the APA protein. Because plants are known to promote post-translational modification such as glycosylation and to be one of the most economic and safe hosts for recombinant protein expression, we have over expressed the APA protein in transgenic tobacco plants aiming to produce a glycosylated version of the protein. Seeds are known to be a well-suited organ to accumulate recombinant proteins, due to low protease activity and higher protein stability. We used a seed-specific promoter from sorghum, a signal peptide to target the protein to the endoplasmic reticulum and ultimately in the protein storage vacuoles. We show that the recombinant protein accumulated in the seeds had similar isoelectric point and molecular weight compared with the native protein. These findings demonstrate the ability of tobacco plants to produce glycosylated APA protein, opening the way for the development of secure, effective and versatile vaccines or therapeutic proteins against tuberculosis.

Highlights

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the first cause of death due to a single infectious agent in the world, leading to approximately 1.8 million deaths annually, and it was estimated that in 2015 there were 10.4 million TB cases globally (WHO 2017)
Production of transgenic plants containing the APA gene The APA gene from M. tuberculosis was amplified by PCR and linked to a synthetic α-coixin signal peptide DNA sequence to enable the targeting of the recombinant protein to the endoplasmic reticulum and accumulation in protein storage vacuoles
The expression cassette was cut from this plasmid and inserted into the plant expression vector pCambia 3301

Summary

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the first cause of death due to a single infectious agent in the world, leading to approximately 1.8 million deaths annually, and it was estimated that in 2015 there were 10.4 million TB cases globally (WHO 2017). Considering the relevance of glycosylation patterns for some proteins and the seek of an efficient and safe production of recombinant proteins without human pathogens in a cost effective way, the use of transgenic plants is a good alternative (Ma et al 2003). The focus of this study was to produce transgenic tobacco plants that express the recombinant protein APA in their seeds For this purpose, we used an expression vector with a seed-specific promoter for γ-kafirin from sorghum, followed by a signal peptide of α-coixin to direct the recombinant protein to the endoplasmic reticulum (for protein glycosylation in the secretory pathway) and consequent accumulation of APA in protein storage vacuoles. We obtained the partially purified recombinant protein and we present evidence of recombinant protein glycosylation due to its affinity for binding to Concanavalin A and due to the presence of multiple isoforms in two-dimensional electrophoresis This open new venues for the production of a recombinant vaccine for tuberculosis

Materials and methods

Results

Discussion