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

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 degrees C for 20 min, but when the temperature exceeded 80 degrees 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.