Molecular Farming in Barley: Development of a Novel Production Platform to Produce Human Antimicrobial Peptide LL-37

Abstract

The peptide LL-37, a component of the human innate immune system, represents a promising drug candidate. In particular, the development of low-cost production platform technology is a critical bottleneck in its use in medicine. In the present study, a viable approach for the LL-37 production in transgenic barley is developed. First, comparative analyses of the effects of different fused peptide epitope tags applicable for accumulation and purification on LL-37 production yield are performed using transient expression in tobacco leaves. Following the selection of the most yielding fusion peptide strategies, eight different constructs for the expression of codon optimized chimeric LL-37 genes in transgenic barley plants are created. The expression of individual constructs is driven either by an endosperm-specific promoter of the barley B1 hordein gene or by the maize ubiquitin promoter. The transgenes are stably integrated into the barley genome and inherited in the subsequent generation. All transgenic lines show normal phenotypes and are fertile. LL-37 accumulated in the barley seeds up to 0.55 mg per 1 kg of grain. The fused epitope tags are cleaved off by the use of enterokinase. Furthermore, in planta produced LL-37 including the fused versions is biologically active.

Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus

“…While challenging due to the physicochemical properties and toxicity of many of these peptides, some of these platforms successfully produce AMPs. These platforms include leaf chloroplasts (Lee et al ., ), rice seeds (Bundó et al ., ; Montesinos et al ., , ), barley seeds (Holásková et al ., ) and hairy roots (Chahardoli et al ., ). However, the yields reported when using these systems were much lower than the AFP yield achieved in N. benthamiana using the TMV‐derived system reported here.…”

“…However, the yields reported when using these systems were much lower than the AFP yield achieved in N. benthamiana using the TMV‐derived system reported here. While our estimated yield of AfpB was approximately 250 μg per gram of fresh N. benthamiana leaf tissue, the reported yields for Retrocyclin‐101 and Protegrin‐1 were ~5 and 8 μg/g of tobacco respectively (Lee et al ., ), those for Cecropin A were ~40 μg/g of rice seeds (Montesinos et al ., ), those for LL37 were ~0.55 μg/g of barley seeds (Holásková et al ., ), and those for Lactoferrin chimera were ~4.8 μg/g of tobacco hairy roots (Chahardoli et al ., ). In addition, all of these plant‐based production systems are based on stable transformation, which makes the process more complex and time consuming.…”

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

“…AMPs apart from exhibiting antimicrobial activity, they were further investigated for inducing resistance against various bacterial and fungal pathogens in plants [ 162 ]. The peptide LL-37 was produced in transgenic barley by expressing codon optimized chimeric LL-37 under the influence of endosperm specific promoter of barley B1 hordein gene, accumulating upto 0.55 mg/kg of grain and the plant-produced LL-37 was biologically active [ 172 ]. An insect antimicrobial peptide, thanatin S, was expressed by fusing with signal peptide of rice Cht1 in Arabidopsis , that showed enhanced resistance to phytopathogenic fungi and bacteria [ 173 ].…”

Molecular Mechanism of Functional Ingredients in Barley to Combat Human Chronic Diseases

“…Human Flt3 ligand isolated from transgenic barley seeds is a glycoprotein including α(1,3)-fucose and α(1,2)xylose, which showed expression of human growth factor in barley grains with active protein [204]. The peptide LL-37 is a component of the human innate immune system, it accumulated 0.55 mg/kg in the barley grains [205]. Human fgf-1 gene was fused with barley α-amylase signal peptide DNA sequence and expressed in transgenic Salvia miltiorrhiza plants driven by 35S promoter; however, recombinant FGF-1 in leaves was 272 ng/ fresh weight [206].…”