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

Int J Mol Sci. 2022 Dec 2;23(23):15202. doi: 10.3390/ijms232315202.

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.

MeSH terms

  • Adhesives / metabolism
  • Anti-Infective Agents* / chemistry
  • Antimicrobial Peptides*
  • Dihydroxyphenylalanine / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Peptides / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Adhesives
  • Anti-Infective Agents
  • Antimicrobial Peptides
  • Dihydroxyphenylalanine
  • Peptides
  • Recombinant Fusion Proteins

Grants and funding

This research was funded by the Intelligent Synthetic Biology Center of the Global Frontier Project, funded by the Ministry of Education, Science and Technology (grant number 2011-0031955); the Development of Industrialization Technology for Crop Virus and Pest Project, funded by the Ministry of Agriculture, Food and Rural Affairs (321109-04-1-SB010); the Center for mRNA/DNA Therapeutics Development and Production Project (P0021532); the Basic Science Research Program (2020M3A9H5104235, NRF-2019R1A2C1090726); the Multi-Department Research and Business Development Program, funded by Sejong city, Republic of Korea (2021-153-0028-0019-002B); and the Research Initiative Program of KRIBB (KGM5402113).