Simple purification of human antimicrobial peptide dermcidin (MDCD-1L) by intein-mediated expression in E.coli

J Microbiol Biotechnol. 2010 Feb;20(2):350-5.

Abstract

Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.

MeSH terms

  • Anti-Bacterial Agents / isolation & purification*
  • Anti-Bacterial Agents / metabolism
  • Anti-Bacterial Agents / pharmacology
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Gene Expression*
  • Humans
  • Inteins*
  • Micrococcus / drug effects
  • Peptides / genetics*
  • Peptides / isolation & purification*
  • Peptides / metabolism
  • Peptides / pharmacology
  • Protein Engineering
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Fusion Proteins / pharmacology

Substances

  • Anti-Bacterial Agents
  • Peptides
  • Recombinant Fusion Proteins
  • dermcidin