Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation

Abstract

Microbial infections remain a global health concern, calling for the urgent need to implement effective prevention measures. Antimicrobial peptides (AMPs) have been extensively studied as potential antimicrobial coating agents. However, an efficient and economical method for AMP production is lacking. Here, we synthesized the direct coating adhesive AMP, NKC-DOPA₅, composed of NKC, a potent AMP, and repeats of the adhesive amino acid 3,4-dihydroxyphenylalanine (DOPA) via an intein-mediated protein ligation strategy. NKC was expressed as a soluble fusion protein His-NKC-GyrA (HNG) in Escherichia coli, comprising an N-terminal 6× His-tag and a C-terminal Mxe GyrA intein. The HNG protein was efficiently produced in a 500-L fermenter, with a titer of 1.63 g/L. The NKC-thioester was released from the purified HNG fusion protein by thiol attack and subsequently ligated with chemically synthesized Cys-DOPA₅. The ligated peptide His-NKC-Cys-DOPA₅ was obtained at a yield of 88.7%. The purified His-NKC-Cys-DOPA₅ possessed surface-binding and antimicrobial properties identical to those of the peptide obtained via solid-phase peptide synthesis. His-NKC-Cys-DOPA₅ can be applied as a practical and functional antimicrobial coating to various materials, such as medical devices and home appliances.

Keywords: Escherichia coli; antimicrobial peptide; intein; intein-mediated protein ligation.

Figure 1

Semi-synthesis of antimicrobial peptides using an intein-mediated protein ligation strategy. The His₆-tag was used for affinity purification, while Mxe GyrA intein was fused to the C-terminus of NKC. The thiol-mediated cleavage of the intein fusion protein using sodium 2-mercaptoethanesulfonate (MESNA) yielded the target peptide with a reactive thioester. The resulting NKC-thioester was mixed with DOPA₅ with an N-terminal cysteine, leading to spontaneous peptide bond formation between the recombinantly obtained NKC-thioester and the chemically synthesized Cys-DOPA₅ via a nucleophilic attack of the N-terminal cysteine on the NKC-thioester.

Figure 2

Expression of the His-NKC-GyrA (HNG) fusion protein. (a) SDS-PAGE and Western blot analyses (anti-His) of HNG expression. Lane M: protein molecular weight marker; Lane U: uninduced E. coli lysate; Lane W: whole cell lysate; Lane S: soluble fraction; Lane I: insoluble fraction. The target protein is indicated by arrows. (b) Analysis of purification of the HNG protein and cleavage activity of GyrA intein. Lane M: protein molecular weight marker; Lane S: soluble fraction; Lane E: elution fraction with 250 mM imidazole; Lane C: product of intein cleavage induced by MESNA. Gyr and HN indicate GyrA intein and His-NKC-thioester, respectively. The green bands on the black background represent the results of Western blot analysis.

Figure 3

Fed-batch fermentation of E. coli BL21 (DE3)-pET21b-HNG. (a) E. coli fed-batch fermentation kinetics for the production of HNG. Cells were cultivated in batch mode until the initially present glucose was depleted. After 5 h, glucose feeding was initiated at a predefined rate. At an OD₆₀₀ of 62, protein expression was induced with 0.1 mM IPTG. Samples were collected to monitor cell OD₆₀₀ (black) and medium glucose content (red). (b) Culture samples collected at induction times of 9, 12, 15, 18, 21, and 24 h were analyzed using SDS-PAGE and Western blotting with an anti-His antibody. Lane M: protein molecular weight marker; Lane W: whole cell lysate; Lane S: soluble fraction; Lane I: insoluble fraction. The target protein is indicated by arrows.

Figure 4

One-pot intein-mediated cleavage and peptide ligation. (a) SDS-PAGE analysis of intein-mediated protein ligation according to TCEP concentration. The reactions were performed using the indicated Cys-DOPA₅ and TCEP concentrations to 0.2 mM HNG. (b) Quantification of the ligated peptide using RP-HPLC (detection at 280 nm). (c) Analysis of intein-mediated protein ligation on an SDS-PAGE gel stained with Coomassie Blue and nitroblue tetrazolium (NBT; purple bands). The reactions were performed at the indicated HNG and Cys-DOPA₅ concentrations. The ligation products show positive NBT staining, indicating the presence of active DOPA. (d) Quantification of the ligated peptide using RP-HPLC (detection at 280 nm).

Figure 5

Comparison of the activity of chemically synthesized His-NKC-Cys-DOPA₅ and semi-synthetic His-NKC-Cys-DOPA₅. (a) Surface-binding activity of peptides. (b) Surface antimicrobial activity against E. coli. The dashed line indicates the number of cells inoculated. Uncoated wells served as controls. CFUs, colony-forming units.