Abstract
ABSTRACTAntimicrobial peptides (AMPs) with their broad and selective antimicrobial activity and lowered risk of resistance development in microbial populations are promining as a new alternative candidate to conventional antibiotics. Plant-based expression systems can be employed as cost-effective and high scale-up capacity production hosts for recombinant expression of AMPs. LFchimera is a chimerical peptide containing LFcin and LFampin antimicrobial peptides of bovine lactoferrin, and it has stronger bactericidal activity. Here, the LFchimera sequence was codon-optimized for tobacco and fused with endoplasmic reticulum retention signals along with a CaMV 35S promoter and then transferred by agrobacterium-mediated transformation. The integration and expression of the transgene in tobacco plants were confirmed by polymerase chain reaction (PCR) and RT-PCR, respectively. Recombinant production of the peptide was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and peptide functional activity was confirmed by the antibacterial evaluation of total protein extracts against various clinical and phytopathogenic bacteria. Finally, LFchimera peptide was partially purified using an affinity column, and the antibacterial activity was shown. Taken together, these results confirmed that tobacco can be utilized for the recombinant production of antimicrobial peptides such as LFchimera.
Highlights
Antimicrobial peptides (AMPs) are considered essential components of the innate immune system, and they typically have broad-spectrum activity against both grampositive and gram-negative bacteria and fungi; their activity extends to enveloped viruses, parasites and cancerous cells [1,2,3,4]
Transgenic tobacco plants carrying a pBI-LFchimera-HIS construct were regenerated on medium containing 100 mg/L kanamycin, and 18 independent kanamycinresistant transgenic lines were selected for further analysis
Integration of the LFchimera gene into the tobacco genome was confirmed by polymerase chain reaction (PCR) amplification using primer pairs specific for LFchimera
Summary
Antimicrobial peptides (AMPs) are considered essential components of the innate immune system, and they typically have broad-spectrum activity against both grampositive and gram-negative bacteria and fungi; their activity extends to enveloped viruses, parasites and cancerous cells [1,2,3,4]. Various AMPs with broad and selective antimicrobial activity and less risk of resistance development in microbial populations are promising, new, alternative candidates for antibiotics [6,7,8,9,10,11]. Several natural AMPs have been identified and isolated from various organisms. Because of the very low AMP concentrations present in most organisms, the direct isolation of AMPs from natural sources to obtain a reasonable amount of active peptide is a time-consuming and high-cost strategy [12]. The chemical synthesis of peptides allows the production of both natural and synthetic AMPs. largescale peptide synthesis is expensive [3]. Largescale peptide synthesis is expensive [3] This method is faced with limitations in terms of complex AMPs that show complex post-translational modifications
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