Transformation strategies for stable expression of complex hetero-multimeric proteins like secretory immunoglobulin A in plants.

Jorge Palaci,Vikram Virdi,Ann Depicker

doi:10.1111/pbi.13098

Abstract

SummaryPlant expression systems have proven to be exceptional in producing high‐value complex polymeric proteins such as secretory IgAs (SIgAs). However, polymeric protein production requires the expression of multiple genes, which can be transformed as single or multiple T‐DNA units to generate stable transgenic plant lines. Here, we evaluated four strategies to stably transform multiple genes and to obtain high expression of all components. Using the in‐seed expression of a simplified secretory IgA (sSIgA) as a reference molecule, we conclude that it is better to spread the genes over two T‐DNAs than to contain them in a single T‐DNA, because of the presence of homologous recombination events and gene silencing. These T‐DNAs can be cotransformed to obtain transgenic plants in one transformation step. However, if time permits, more transformants with high production levels of the polymeric protein can be obtained either by sequential transformation or by in‐parallel transformation followed by crossing of transformants independently selected for excellent expression of the genes in each T‐DNA.

Highlights

Secretory immunoglobulin A (SIgA) is a heterodecameric Ig of 400 kDa that is predominantly involved in the protection of the mucosal surfaces against pathogenic infections
The few milligram production levels of SIgAs attained in mammalian or insect cell fermenters are not sufficient or cost effective to be translated into the clinic
The J-chain, secretory component (SC) and VHH-IgA encoding sequences were each fused with the N-terminal 2S2 signal peptide and the Cterminal KDEL motif sequences for ER targeting and retention, respectively

Summary

Introduction

Secretory immunoglobulin A (SIgA) is a heterodecameric Ig of 400 kDa that is predominantly involved in the protection of the mucosal surfaces against pathogenic infections. Application of pathogen-specific SIgAs at the mucosal surfaces can provide immediate protection (Corthesy, 2010) but requires bulk amounts of the SIgA protein in the range of gram amounts per day and per patient. Of the expression systems evaluated, plants have been shown to be the most successful platform for SIgAs (Ariaans et al, 2014; Ma et al, 1995). The bacterial and yeast systems currently do not efficiently secrete assembled Igs, and the levels of correctly post-transcriptionally modified SIgAs are too low (Fischer et al, 2004; Vasilev et al, 2016). Several different SIgAs have been expressed successfully in plants, and one of these SIgAs, that is, CaroRx, has been approved for use as medical device for dental care (Paul and Ma, 2011)

Results

Discussion

Conclusion