Method for Analyzing the Antimicrobial Activity of Peptides via Escherichia coli Expression System

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Abstract

The test system for an assay of new potential antimicrobial peptides (AMP) based on the expression of recombinant AMP-encoding genes in Escherichia coli cells has been proposed. This method has a number of advantages over the use of chemically synthesized peptides and both approaches effectively complement each other. Our approach does not impose limitations on the AMP size, facilitates high-throughput screening of mutant plasmid libraries, and has lower cost and complexity compared to the use of synthetic peptides. The core of our methodology involves transformation of the model gram-negative bacterium E. coli with plasmids carrying a recombinant AMP-encoding gene regulated by an inducible promoter. Following transcription induction, bacteria synthesize the AMP, which ultimately leads to cell death. The assessment of bacterial growth is carried out either by measuring the optical density of a bacterial culture grown in liquid media in a microplate or by drip seeding of serial culture dilutions on an agar-based nutrient medium.

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About the authors

E. N. Grafskaia

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency

Author for correspondence.
Email: grafskayacath@gmail.com
Russian Federation, Moscow, 119435

D. D. Kharlampieva

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency

Email: grafskayacath@gmail.com
Russian Federation, Moscow, 119435

P. A. Bobrovsky

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency; Federal State Autonomous Educational Institution of Higher Education “Moscow Institute of Physics and Technology (National Research University)”

Email: grafskayacath@gmail.com
Russian Federation, Moscow, 119435; Dolgoprudny, 141701

M. Y. Serebrennikova

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency; Federal State Autonomous Educational Institution of Higher Education “Moscow Institute of Physics and Technology (National Research University)”

Email: grafskayacath@gmail.com
Russian Federation, Moscow, 119435; Dolgoprudny, 141701

V. N. Lazarev

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency; Federal State Autonomous Educational Institution of Higher Education “Moscow Institute of Physics and Technology (National Research University)”

Email: grafskayacath@gmail.com
Russian Federation, Moscow, 119435; Dolgoprudny, 141701

V. A. Manuvera

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency; Federal State Autonomous Educational Institution of Higher Education “Moscow Institute of Physics and Technology (National Research University)”

Email: grafskayacath@gmail.com
Russian Federation, Moscow, 119435; Dolgoprudny, 141701

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2. 1. Optical densities of E. coli BL21-gold (DE3) cell cultures transformed by AMP-encoding expression plasmids after 24 h incubation. Plasmids encode AMP without the signal peptide (a), plasmids encode AMP with the signal peptide pelB (b). K – control cells of E. coli BL21-gold (DE3) transformed by recipient plasmids without the coding AMP insert; Mel, Cecr, Apid, HmAmp2 and HmAmp4 — E. coli BL21-gold (DE3), transformed by a plasmid encoding melittin, cecropin, apidacin, HmAmp2 and HmAmp4, respectively; 1 — LB, medium without a transcription inducer; 2 — LB + IPTG, LB medium with 0.1 mM IPTG inductor.

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3. 2. Growth curves of E. coli BL21-gold (DE3) cultures transformed by expression plasmids encoding AMP with the pelB signaling peptide. a – E. coli BL21-gold (DE3) control cells transformed by the pET-22 plasmid(b); Mel (b), Cecr(c), Apid (d), HmAmp2 (e) and HmAmp4 (e) — E. coli BL21-gold (DE3) cells transformed by the plasmid, encoding melittin, cecropin, apidacin, HmAmp2 and HmAmp4 fused with the signal peptide, respectively. 1 — LB without a transcription inducer; 2 — LB + IPTG, medium with 0.1 mM IPTG inducer.

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4. 3. Photographs of Petri dishes after drip seeding of serial dilutions (10-105) of bacterial cultures and incubation for 16 hours at 37 °C. K – control cells of E. coli BL21-gold (DE3) transformed by plasmid pET-22(b); Mel, Cecr, Apid, HmAmp2 and HmAmp4 – E. coli BL21-gold (DE3) cells transformed by plasmid encoding melittin, cecropin, apidacin, HmAmp2 and HmAmp4 fused with the signal peptide pelB Accordingly, LB is a medium without a transcription inducer; LB + IPTG is a medium with 0.1 mM IPTG inducer. The scale of bacterial culture breeding is shown above.

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