Expression, Purification, and Characterization of a Novel Hybrid Peptide with Potent Antibacterial Activity

Abstract

The hybrid peptide cecropin A (1⁻8)⁻LL37 (17⁻30) (C⁻L), derived from the sequence of cecropin A (C) and LL-37 (L), showed significantly increased antibacterial activity and minimized hemolytic activity than C and L alone. To obtain high-level production of C⁻L, the deoxyribonucleic acid sequence encoding C⁻L with preferred codons was cloned into pET-SUMO to construct a fusion expression vector, and overexpressed in Escherichia coli (E. coli) BL21 (DE3). The maximum fusion protein (92% purity) was obtained with the yield of 89.14 mg/L fermentation culture after purification with Ni-NTA Sepharose column. The hybrid C⁻L was cleaved from the fusion protein by SUMO-protease, and 17.54 mg/L pure active C⁻L was obtained. Furthermore, the purified C⁻L showed identical antibacterial and hemolytic activity to synthesized C⁻L. Stability analysis results exhibited that the activity of C⁻L changed little below 80 °C for 20 min, but when the temperature exceeded 80 °C, a significant decrease was observed. Varying the pH from 5.0 to 10.0 did not appear to influence the activity of C⁻L, however, pH below 4.0 decreased the antibacterial activity of C⁻L rapidly. Under the challenge of several proteases (pepsin, trypsin, and proteinase K), the functional activity of C⁻L was maintained over 50%. In summary, this study not only supplied an effective approach for high-level production of hybrid peptide C⁻L, but paved the way for its further exploration in controlling infectious diseases of farm animals or even humans.

Keywords: Escherichia coli; antibacterial activity; fusion expression; hybrid peptide; stability.

Figure 1

The schematic of construction of expression vector.

Figure 2

Tricine-SDS-PAGE analysis of supernatant with 1.5 mM IPTG induction. Lane 1, supernatant of non-induced E. coli BL21 (DE3); Lane 2, 3, 4, 5, 6 were supernatant of 1.5 mM IPTG-induced E. coli BL21 (DE3) for 1, 2, 3, 4, 5 h respectively; Lane M, marker. The arrows indicated fusion protein SUMO–C–L.

Figure 3

Tricine-SDS-PAGE analysis of purified fusion protein SUMO–C–L. Lane M, the molecular weight of marker; Lane 1, the supernatant of IPTG-induced E. coli BL21 (DE3); Lane 2, the supernatant of non-induced bacterial lysate; Lane 3, the purified fusion protein SUMO–C–L, arrow in the lane indicated the fusion protein SUMO–C–L; Lane 4, the enzymatic reaction solution of fusion protein, arrows in the lane were SUMO and recombinant C–L.

Figure 4

Tricine-SDS-PAGE and antibacterial analysis of purified recombinant C–L. Lane M, the molecular weight of marker; Lane 1, the purified recombinant C–L, arrow in the lane indicated the recombinant C–L.

Figure 5

MALDI-TOF/TOF MS mass spectra of purified C–L.

Figure 6

The antimicrobial activity of recombinant C–L against S. aureus ATCC25923. A, recombinant C–L (2 μg); B, fusion SUMO–C–L (10 μg); C, the negative control, sodium phosphate buffer (PBS).

Figure 7

Effects of temperature (A), pH (B), and proteases (C) on recombinant (left) and synthesized C–L (right). (A) The peptide sample kept at 4 °C was used as a control. (B) The sample kept in the original culture (pH 6.0) was used as a control. (C) The original sample without any enzymatic treatment was used as a control. The graphs were derived from average values for three replicate experiments and error bars show standard deviations. E. coli ATCC25923 was used as the indicator strain.