Recombinant production of bovine Lactoferrin-derived antimicrobial peptide in tobacco hairy roots expression system

Chahardoli, Mahmood; Fazeli, Arash; Ghabooli, Mehdi

doi:10.1016/j.plaphy.2017.12.037

Cited by 33 publications

(28 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, we successfully expressed and accumulated LFchimera peptide by recombinant production in an in vitro plant culture system and tobacco hairy roots [39]. In this study, our efforts have focused on the use of tobacco whole plants as an expression host for largescale and cost-effective production of LFchimera and to overcome the limitations of in vitro expression systems.…”

Section: Introductionmentioning

confidence: 99%

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

Chahardoli

Fazeli

Niazi‎

et al. 2018

Biotechnology & Biotechnological Equipment

Self Cite

View full text Add to dashboard Cite

Antimicrobial 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 sulphatepolyacrylamide 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.

show abstract

Section: Introductionmentioning

confidence: 99%

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

Chahardoli

Fazeli

Niazi‎

et al. 2018

Biotechnology & Biotechnological Equipment

Self Cite

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Discussionmentioning

confidence: 97%

“…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 lg per gram of fresh N. benthamiana leaf tissue, the reported yields for Retrocyclin-101 and Protegrin-1 were~5 and 8 lg/g of tobacco respectively (Lee et al, 2011), those for Cecropin A were~40 lg/ g of rice seeds (Montesinos et al, 2016), those for LL37 werẽ 0.55 lg/g of barley seeds (Hol askov a et al, 2018), and those for Lactoferrin chimera were~4.8 lg/g of tobacco hairy roots (Chahardoli et al, 2018). In addition, all of these plant-based production systems are based on stable transformation, which makes the process more complex and time consuming.…”

Section: Discussionmentioning

confidence: 99%

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

Shi

Cordero

Garrigues

et al. 2018

Plant Biotechnology Journal

View full text Add to dashboard Cite

Summary Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins ( AFP s) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus ( TMV ). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFP s in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFP s were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFP s fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.

show abstract

“…Analysis of total proteins extracted from transgenic HRs indicated a strong antimicrobial activity against both gram‐positive and gram‐negative bacteria as well as pathogenic fungi (Moghadam et al, ). Chahardoli, Fazeli, and Ghabooli () expressed a lactoferricin encoding protein in tobacco HR culture with potent antibacterial activity against Escherichia coli (Chahardoli et al, ). Introduction of ranalexin peptide in tobacco HRs resulted in efficient production of ranalaxin in HRs with strong inhibitory effect on multidrug resistant gram‐positive and gram‐negative pathogens such as Staphylococcus aureus , Streptococcus pyogenes , and E. coli and on Enterococcus strains resistant to vancomycin, suggesting that tobacco HR culture was a suitable system to produce ranalexin and other recombinant peptides (Aleinein et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…Analysis of total proteins extracted from transgenic HRs indicated a strong antimicrobial activity against both gram-positive and gram-negative bacteria as well as pathogenic fungi (Moghadam et al, 2016). Chahardoli, Fazeli, and Ghabooli (2018) expressed a lactoferricin encoding protein in tobacco HR culture with potent antibacterial activity against Escherichia coli (Chahardoli et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco

et al. 2019

View full text Add to dashboard Cite

Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin‐binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD‐DrsB1 and pGSA/DrsB1‐CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi‐quantitative RT‐PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD‐DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1‐CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.

show abstract

Recombinant production of bovine Lactoferrin-derived antimicrobial peptide in tobacco hairy roots expression system

Cited by 33 publications

References 46 publications

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

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

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

Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco

Contact Info

Product

Resources

About