High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli

Abstract

Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34-39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production.

Keywords: antimicrobial peptide; cecropin-like; expression; intein; self-cleavage.

Figure 1

Gene map of the recombinant plasmid pET11b-His6-intein-KR12AGPWR6. (A) The illustration of inserted His6-intein-KR12AGPWR6 sequence. (B) Optimized DNA sequence and codon map. His6-tag are in bold; the sequence of intein is underlined; the sequence of KR12AGPWR6 is shaded.

Figure 2

SDS-PAGE analysis of recombinant His6-Intein-KR12AGPWR6 in E. coli BL21. (A) SDS-PAGE of recombinant His6-Intein-KR12AGPWR6 expressed in E. coli BL21 with or without 0.4 mM IPTG at 20 °C for 4, 8, 24 h. Lane 1: protein molecular weight markers (kDa). Lane 2: before induction. Lanes 3–5: with IPTG induction at 20 °C for 4, 8, 24 h. Lanes 6–8: without IPTG induction at 20 °C for 4, 8, 24 h. (B) Relative intensities of the His6-Intein-KR12AGPWR6 bands. Proteins were visualized using Coomassie blue staining.

Figure 3

SDS-PAGE analysis of recombinant His6-Intein-KR12AGPWR6 expressed in E. coli at different steps of purification procedure. Lane 1: protein molecular weight markers (kDa); lane 2: supernatant of cell lysate; lane 3: flow through; lane 4–6: washing by 40 mM imidazole; lane 7–10: elution by 400 mM imidazole. Proteins were visualized using Coomassie blue staining.

Figure 4

SDS-PAGE analysis of pH, temperature, and time optimization for intein’s self-cleavage. (A) SDS-PAGE analysis of pH optimization for intein’s self-cleavage. Lane 1: protein molecular weight markers (kDa); lane 2: protein in pH 10 buffer at 4 °C before self-cleavage; lanes 3–5: protein in different pH at 55 °C for 18 h; lanes 6–8: protein in different pH at 55 °C for 72 h; lane 9: the supernatant of protein in pH 10 at 55 °C for 72 h; lane 10: chemical synthesized sKR12AGPWR6 was used as a control. (B) Quantification of intein self-cleavage rates. (C) SDS-PAGE analysis of temperature optimization for intein’s self-cleavage. Lane 1: protein molecular weight markers (kDa). Lane 2 and 3: the targeted proteins were cleaved in pH 10 buffer for 18 h at 4 °C and 37 °C, respectively. Lane 4 and 5: targeted proteins were cleaved in pH 10 buffer for 72 h at 4 °C and 37 °C, respectively. Lane 6: synthetic KR12AGPWR6 was used as a control. (D) Quantification of intein self-cleavage rates. Proteins were observed using Coomassie blue staining.

Figure 5

Reversed-phase HPLC purification of KR12AGPWR6 after intein’s self-cleavage. The samples were resuspended with 6 M guanidine hydrochloride and purified by reversed-phase HPLC on the Prominence HPLC System equipped with a C18 column. The column was equilibrated with ddH₂O containing 0.1% (v/v) trifluoroacetic acid (TFA) and the gradient ranging from 15 to 100% (v/v) methanol containing 0.1% (v/v) TFA for 75 min at a flow rate of 1 mL/min. Signals were detected by UV 280 nm.

Figure 6

Mass analysis of KR12AGPWR6 from RP-HPLC. The molecular weight of the purified KR12AGPWR6 was found to be 2823.664 Da. The theoretical MW of KR12AGPWR6 was calculated to be 2824.3 Da.

Figure 7

Circular dichroism spectra of synthetic and recombinant KR12AGPWR6 in different environments. Circular dichroism spectra of 60 µM synthetic and recombinant KR12AGPWR6 in 30% TFE buffer in pH7.4 at 25 °C.

Figure 8

¹H-¹⁵N HSQC spectra of 0.6 mM rKR12AGPWR6 in 20 mM sodium phosphate buffer, pH 4.5, 298 K. The ¹⁵N-labeled samples were expressed in E. coli BL21 (DE3) cells grown in M9 medium containing ¹⁵N-labeled ammonium chloride (1 g/L), and the purification of peptides as mentioned above. The cross peaks of rKR12AGPWR6 were shown in red.