Transient Expression of Recombinant Antimicrobial Peptide Meucin18 in Nicotiana tabacum and its Antimicrobial Susceptibility Testing
Introduction: Scorpion venom peptides as antimicrobial peptides (AMPs) possess high bactericidal activity against a broad range of Gram-positive and Gram-negative bacteria. Meucin-18, a venom peptide of molecular weight 2.1 kDa from Mesobuthus eupeus, shows high bacteriolytic potential, and so recom...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-03-01
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| Series: | International Journal of Infectious Diseases |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1201971224004818 |
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| Summary: | Introduction: Scorpion venom peptides as antimicrobial peptides (AMPs) possess high bactericidal activity against a broad range of Gram-positive and Gram-negative bacteria. Meucin-18, a venom peptide of molecular weight 2.1 kDa from Mesobuthus eupeus, shows high bacteriolytic potential, and so recombinant production in E. coli is highly unlikely. Hence, recombinant Meucin-18 is transiently expressed in an efficient alternate host, Nicotiana tabacum plant system, to test its antibacterial efficacy. Methods: The gene sequence (54 bp) coding Meucin18 fused with a C-terminal 6X histidine tag and an ER localization signal KDEL, was codon optimized, synthesized de novo and custom cloned into pENTR-D-TOPO Gateway vector. Gateway LR cloning was performed to produce expression construct pEAQ-HT-DEST3-Meucin18. A quantity of 100 ng expression construct was transformed into electrocompetent Agrobacterium tumefaciens GV3101 through electroporation, pulsed at 1800V for 5 ms. Agrosuspension was prepared with 1X MES infiltration medium containing 100 µM acetosyringone. Leaves of N. tabacum plants maintained at 26°C were syringe infiltrated with agrosuspension in abaxial side. Total protein was extracted from 5 days post infiltration (dpi) leaves using extraction buffer (Tris-HCl 100mM; pH 7.5 and 0.1M KH2PO4; pH 6.4). Recombinant Meucin18 AMP was purified by Ni-NTA affinity chromatography and analyzed on tricine-SDS-PAGE, and Western blot using anti-His6 peroxidase antibody. Recombinant fusion AMP will be tested for its efficacy against bacteria (pathogenic and non-pathogenic) using classical agar diffusion and broth microdilution assays. Results: Plant expression construct pEAQ-HT-DEST3-Meucin18 was transformed into E. coli DH5ɑ for propagation and confirmed through PCR and restriction digestion analysis. pEAQ-HT-DEST3-Meucin18 from A. tumefaciens GV3101 was isolated and confirmed using PCR analysis. Total crude protein from 5 dpi N. tabacum leaf sample was measured, at A280 using an Epoch Take3 spectrophotometer, to be 11.51 mg/g and 20.76 mg/g of fresh leaf tissue and the protein content from uninfiltrated leaf sample was measured to be 8.90 mg/g of fresh leaf tissue. The quantity of purified sample was found to be 21.4 µg/g that will be confirmed using 16% tricine-SDS-PAGE and Western blot. Further, recombinant Meucin18 will be tested for its bactericidal potential using agar diffusion and broth microdilution assays. Discussion: Scorpion venom peptides have been assessed for their potential to act against a broad range of bacteria including Methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Bacillus subtilis, Micrococcus luteus, Pseudomonas aeruginosa and E. coli. N. tabacum plant system proves to be a better host system for transient expression of recombinant functional antimicrobial peptides such as protegrin-1. Further optimization, extraction and purification of Meucin18 from N. tabacum will be useful in the development of a broad-spectrum antibacterial laboratory reagent. Conclusion: The purified recombinant AMP Meucin18 from N. tabacum will be an efficient antibacterial agent that could be used against a broad range of bacteria. |
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| ISSN: | 1201-9712 |