Optimization of the human colorectal carcinoma antigen GA733-2 production in tobacco plants

Abstract

The colorectal carcinoma-associated protein GA733-2 is one of the representative candidate protein for the development of plant-derived colorectal cancer vaccine. Despite of its significant importance for colorectal vaccine development, low efficiency of GA733-2 production limits its wide applications. To improve productivity of GA733-2 in plants, we here tested multiple factors that affect expression of recombinant GA733-2 (rGA733-2) and rGA733 fused to fragment crystallizable (Fc) domain (rGA733-Fc) protein. The rGA733-2 and rGA733-Fc proteins were highly expressed when the pBINPLUS vector system was used for transient expression in tobacco plants. In addition, the length of interval between rGA733-2 and left border of T-DNA affected the expression of rGA733 protein. Transient expression analysis using various combinations of Agrobacterium tumefaciens strains (C58C1, LBA4404, and GV3101) and tobacco species (Nicotiana tabacum cv. Xanthi nc and Nicotiana benthamiana) revealed that higher accumulation of rGA733-2 and rGA733-Fc proteins were obtained by combination of A. tumefaciens LBA4404 and Nicotiana benthamiana. Transgenic plants generated by introduction of the rGA733-2 and rGA733-Fc expression cassettes also significantly accumulated corresponding recombinant proteins. Bioactivity and stability of the plant-derived rGA733 and rGA733-Fc were evaluated by further in vitro assay, western blot and N-glycosylation analysis. Collectively, we here suggest the optimal condition for efficient production of functional rGA733-2 protein in tobacco system.

Immunotherapeutic effects of recombinant colorectal cancer antigen produced in tomato fruits

“…To generate transgenic tomato plants that stably express rGA733-Fc, a plant expression vector that expresses rGA733-Fc fused with ER-targeting signals and ER retention motif under the control of the 35S promoter (pBINPLUS-GA733-Fc) 41 were transformed into Solanum lycopersicum cv. Micro-Toms plants using Agrobacterium -mediated genetic transformation (Fig.…”

“…However, low production rate of GA733-2 in plants is regarded as a limitation for development of edible colorectal plant vaccine. Several approaches have been devised to increase GA733-2 production in plants 40 , 41 . Among them, fusion of GA733-2 with fragment crystallizable (Fc) domain and ER retention peptide (rGA733-Fc) greatly increased GA733-2 production rate 40 , 41 .…”

“…Several approaches have been devised to increase GA733-2 production in plants 40 , 41 . Among them, fusion of GA733-2 with fragment crystallizable (Fc) domain and ER retention peptide (rGA733-Fc) greatly increased GA733-2 production rate 40 , 41 . In addition, the rGA733-Fc recombinant protein successfully induced apoptosis and reduced tumorigenicity of MC38 and HCT116 carcinomal cells 41 .…”

“…Among them, fusion of GA733-2 with fragment crystallizable (Fc) domain and ER retention peptide (rGA733-Fc) greatly increased GA733-2 production rate 40 , 41 . In addition, the rGA733-Fc recombinant protein successfully induced apoptosis and reduced tumorigenicity of MC38 and HCT116 carcinomal cells 41 . In this study, we attempted to generate transgenic tomato plant that expresses rGA733-Fc in its fruits, and determine its effects on colorectal cancer development using mice system.…”

Biotechnological Insights on the Expression and Production of Antimicrobial Peptides in Plants

“…An early study of pharmaceutical recombinant antibodies stably integrated and produced in transgenic tobacco plants was recorded in 1989. Since then, several proteins have been produced in stably transformed plants, including anti-cancer agents [ 143 , 144 ] and antimicrobial peptides as anti-infectives [ 145 ]. AMPs with anti-bacterial or anti-fungal properties can be stably expressed in plants which confers disease resistance against plant pathogens which in turn increases the yield, quality and safety of agricultural products.…”