Recombinant expression and biological characterization of the antimicrobial peptide fowlicidin-2 in Pichia pastoris

Abstract

Fowlicidins are a group of cathelicidin antimicrobial peptides that were initially identified in chickens. Fowlicidin-2, which is composed of 31 amino acids, is widely expressed in the majority of tissues in chickens and has an important role in innate immunity. In the present study, a recombinant expression system for fowlicidin-2 was successfully constructed using Pichia pastoris X-33 and the expression vector pPICZα-A. Under the optimized fermentation conditions, 85.6 mg fowlicidin-2 with >95% purity was obtained from 1 liter culture medium following purification by ion exchange chromatography and reversed phase high performance liquid chromatography. The recombinant fowlicidin-2 exhibited broad spectrum antimicrobial activity and had a minimum inhibitory concentration ranging from 1 to 4 µM. Furthermore, recombinant fowlicidin-2 exhibited hemolytic activity, promoting 50% human erythrocyte hemolysis in the concentration range of 128-256 µM, and anticancer activity, resulting in the death of 50% of A375 human malignant melanoma cells in the concentration range of 2-4 µM. The results of the present study suggest that recombinant fowlicidin-2 may be a promising candidate for therapeutic applications.

Keywords: Pichia pastoris; antibacterial activity; anticancer activity; antimicrobial peptides; fowlicidin-2; recombinant expression.

Figure 1.

Nucleotide sequence of DNA encoding fowlicidin-2 and the C-terminal amino acids of the α-factor signal peptide. The Kex2 and Ste13 cleavage sites for proteolytic processing and α-factor-driven secretion of the fusion protein are indicated by arrowheads. The synthesized DNA fragment is shown in bold. The sequences of EcoRI and XbaI sites are italicized. The amino-acid and encoding sequences of fowlicidin-2 are underlined. Kex2, Kexin protease 2; Ste13, dipeptidyl aminopeptidase.

Figure 2.

Polymerase chain reaction (PCR) screening of the recombinant pPICZα-A-fowlicidin-2 expression plasmid. Lane M contains the DNA molecular weight marker, lanes 1 and 2 contain the PCR product of the pPICZα-A-fowlicidin-2 plasmid, lane 3 contains the PCR product of the pPICZα-A plasmid, and lane 4 is the negative control.

Figure 3.

Tricine-sodium dodecyl sulfate-polyacrylamide gel analysis of the supernatant from fermentation broth containing secreted recombinant fowlicidin-2. Lane M contains the protein molecular weight marker, and lanes 1–7 contain the medium from cultures of fowlicidin-2-expressing Pichia pastoris at 12, 24, 36, 48, 60, 72 and 84 h following methanol induction, respectively.

Figure 4.

Tricine-sodium dodecyl sulfate-polyacrylamide gel analysis of the purified recombinant (r)fowlicidin-2. Lane M contains the protein molecular weight marker, and lanes 1 and 2 contain the purified rfowlicidin-2 from different batches.

Figure 5.

Hemolytic activity of various concentrations of recombinant fowlicidin-2 on human red blood cells.

Figure 6.

Anticancer activity of various concentrations of recombinant fowlicidin-2 against A375 human malignant melanoma cells, RAW264.7 mouse macrophages and chick embryo fibroblasts.