Effect of Amino Acid Substitutions on Biological Activity of Antimicrobial Peptide: Design, Recombinant Production, and Biological Activity

Abstract

Recently, antimicrobial peptides have been introduced as potent antibiotics with a wide range of antimicrobial activities. They have also exhibited other biological activities, including anti-inflammatory, growth stimulating, and anti-cancer activities. In this study, an analog of Magainin II was designed and produced as a recombinant fusion protein. The designed sequence contained 24 amino acid residues (P24), in which Lys, His, Ser residues were substituted with Arg and also, hydrophobic Phe was replaced with Trp. Recombinant production of P24 in Escherichia coli (E. coli) BL21 using pTYB21, containing chitin binding domain and intein sequence at the N-terminus of the peptide gene, resulted in 1 μg mL^-1 product from culture. Chitin column chromatography, followed by online peptide cleavage with thiol reducing agent was applied to purify the peptide. Antimicrobial activity was evaluated using five bacteria strains including Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumonia, E. coli, and Pseudomonas aeruginosa. Designed AMP exhibited promising antimicrobial activities with low minimum inhibitory concentration, in the range of 64-256 µg/mL. P24 showed potent antimicrobial activity preferably against Gram-positive bacteria, and more potent than pexiganan as a successful Magainin II analog for topical infections. In general, further modification can be applied to improve its therapeutic index.

Keywords: Amino acid substitution; Antimicrobial peptide; Chitin binding domain; Intein linker; Recombinant production.

Figure 1

The structure of recombinant vector pTYB21 for novel designed AMP (P24). (A) The structure of pTYB21; the important parts are outlined with red box. (B) The sequence of first 5 amino acids at MCS of pTYB21, was added to the N-terminal of P19, indicated in red box

Figure 2

(A) Predicted structure of designed peptide P24 by PepFold ((http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD/) with two distinct helical structures; (B) Helical Wheel structure of designed peptide P24 with obvious amphipaticity; green: most hydrophobicity, yellow color: least hydrophobicity, circle: hydrophil amino acids, pentagonal: positive charge amino acids, triangle: negative charge amino acids. Evaluated by Helical Wheel Projections from RZ Lab (http://rzlab.ucr.edu/scripts/ wheel/wheel.cgi?submit)

Figure 3

Electrophoresis evaluation of PCR products on agarose gel 2%: 1) ladder 100bp-10 kbp 2) PCR products of selected colons

Figure 4

Electrophoretic analysis on 15% SDS–PAGE (A) of intein-fusion expression in E. coli BL21. Lane 1, Low molecular weight protein ladder; Lane 2, before IPTG induction (un-induced); Lane 3, 1 h after IPTG induction; Lane 4, 3 h after induction; Lane 5, 4 h after induction; (B) samples obtained from different steps of purification. Lane 1: protein ladder, lane 2: sample after solubilization of inclusion bodies loaded on chitin column, lane 3: samples after first wash, lane 4: sample after second wash, lane 5: purified peptide after cleavage by DTT