Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag

Abstract

Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in Escherichia coli and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Burkholderia cenocepacia strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for Pseudomonas aeruginosa) were demonstrated to be susceptible to ABP-CM4, including a resistant Burkholderia cenocepacia clinical strain. As for Synoeca-MP, although it did not inhibit the growth of Pseudomonas aeruginosa or Klebsiella pneumoniae, it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria.

Antiviral Peptides in Antimicrobial Surface Coatings—From Current Techniques to Potential Applications

“…In recombinant peptide production, a modified peptide encoded by recombinant DNA is cloned in a foreign expression system. Recombinant production gives more control over the peptide sequence, higher yields, and is a more sustainable source of the peptides [ 78 ]. However, this technique requires initial work on proper plasmid construction and genetic design.…”

“…These materials contained antimicrobial peptide sequences. They also concluded that the antimicrobial efficacy of these novel materials is attributable to the peptide sequence, not to the backbone polymer structure [ 78 ]. De Costa et al developed a new fusion material, BMAP-18-A200,based film [ 153 ].…”

“… ( a ) Sources of antiviral peptides (reprinted with permission from Agarwal et al, 2021 [ 77 ]); ( b ) summary of recombinant antimicrobial peptide production steps. ELR is an elastin-like recombinamer used as a fusion tag to purify the recombinant peptide in the downstream application (reprinted with permission from Pereira et al, 2021 [ 78 ]). …”

Smart tools for antimicrobial peptides expression and application: The elastic perspective

“…Thus, ELP was used as the purification tag as in the previous examples. However, in this case, after the chemical cleavage and the ITC purification, a chromatographic step was required to improve the recovery of the AMPs (Pereira et al, 2021). This approach showed a yield about 2‐times higher for the Moricin CM4, relative to that obtained with the ELP‐intein‐AMP fusion setup (Table 1, #2 and Figure 2a).…”

Synoeca‐MP: New insights into its mechanism of action by using NMR and molecular dynamics simulations approach

“…[ 20 ] Due to its low hemolytic and cytotoxic activities, several biotechnological applications have been used to obtain efficient and cost‐effective approaches for the recombinant production and purification of this peptide. [ 21 ]…”