Transient Expression of Recombinant Antimicrobial Peptide Meucin18 in Nicotiana tabacum and its Antimicrobial Susceptibility Testing

Abstract

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.