Enhanced Expression of B-Subunit of <I>Escherichia coli</I> Heat-Labile Enterotoxin in Tobacco by Optimization of Coding Sequence

Abstract

Escherichia coli heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant for coadministered antigens. We synthesized a gene encoding the B-subunit of LT (LTB) adapted to the coding sequence of tobacco plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its level of expression in plants. The synthetic LTB gene was cloned into a plant expression vector adjacent to the CaMV 35S promoter and was introduced into tobacco by Agrobacterium-mediated transformation. The amount of LTB protein detected in transgenic tobacco leaves was 2.2% of the total soluble plant protein, which is approx 200-fold higher than in previous reports of native LTB gene expression in transgenic plants. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers.

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

“…This increases the translational efficiency and subsequently improves the recombinant protein expression and the overall costs of protein production in the considered host [60,61]. Codon optimization is employed to increase the production of human and animal recombinant proteins in the tobacco expression system [49,57].…”

“…In this regard and because of the mammalian origin of LFchimera, genetic codes for 20 out of the 34 amino acids of the LFchimera peptide sequence were codonoptimized for expression in tobacco whole plant, and the translational start-site of the heterologous peptide was modified to match the Kozak consensus for efficient initiation of translation in plants (Figure 1) [61,63]. The results from RT-PCR showed that the LFchimera was properly transcribed, and the transcripts were stable in the plants.…”

Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae

“…After blocking, the filter was incubated with either anti-LTB antiserum (Immunology Consultants Laboratory, Inc.) or anti-ApxIIA antiserum (26) then was bound to anti-rabbit IgG or anti-mouse IgG, respectively, conjugated to alkaline phosphatase as a secondary antibody (Promega). The color was developed using 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (BCIP-NBT) (USB) in TMN buffer (100 mM Tris [pH 9.5], 5 mM MgCl 2 , and 100 mM NaCl), according to the methods described by Kang et al (22).…”

“…The plate was read at 405 nm in an ELISA reader (Packard Instrument MRA-006) and quantified via comparison with a known quantity of purified bacterial LTB. The purified bacterial LTB, obtained from the previous study (22), was serially diluted within the range of the concentrations from 0.1 ng to 10 ng. GM1 ganglioside binding assay.…”

Current status in molecular farming

“…planta-pharma, plant bioreactors, plant biofactory, 그리고 pharmaceutical gardening 등 많은 용어들이 비슷한 의미로 혼용하여 사용하고 있다 (Basaran and Rodriguez-Cerezo 2008 (Mason et al 1998;Kang et al 2004), 유전자의 안정성에 영향을 미 치는 요소 제거 (De Jaeger et al 2002;Ibrahim et al 2001;Outchkourov et al 2003) (Barta et al 1986;De Zoeten et al 1989;Sijmons et al 1990), 1989년에는 담배의 잎에서 항체를 발현을 시킴으로서 식물에서 복잡 한 형태의 치료용 동물단백질을 생산할 수 있다는 가능성 이 제시되었다 (Hiatt et al 1989 (Poirier et al 1992).…”