Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies

Yifeng Li, Xia Li, Guangshun Wang

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

Antimicrobial peptide LL-37 plays an important role in human body's first line of defense against infection. To better understand the mechanism of action, it is critical to elucidate the three-dimensional structure of LL-37 in complex with bacterial membranes. We present a bacterial expression system that allows the incorporation of 15N and other isotopes into the polypeptide for nuclear magnetic resonance (NMR) analysis. The DNA sequence encoding full-length LL-37 was chemically synthesized and cloned into the pET-32a(+) vector for protein expression in Escherichia coli strain BL21(DE3). The peptide was expressed directly as a His-tagged fusion protein without the inclusion of its precursor sequence. LL-37 was released from the fusion by formic acid cleavage at the AspPro dipeptide bond and separated from the carrier thioredoxin by affinity chromatography and reverse-phase HPLC. The peptide was identified by polyacrylamide gel electrophoresis and further confirmed by mass spectrometry and NMR spectroscopy. Antibacterial activity assays showed that the recombinant LL-37 purified from the bacterial source is as active as that from chemical synthesis. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 111 peptides contain a Met residue, but only 5 contain the AspPro pair, indicating a broader application of formic acid than cyanogen bromide in cleaving fusion proteins. The successful application to the expression of the 66-residue cytoplasmic tail of human MUC1 indicates that the system can be applied to other peptides as well.

Original languageEnglish (US)
Pages (from-to)498-505
Number of pages8
JournalProtein Expression and Purification
Volume47
Issue number2
DOIs
StatePublished - Jun 2006

Keywords

  • Antimicrobial peptide
  • Chemical cleavage
  • Escherichia coli
  • Formic acid
  • Isotope labeling
  • LL-37
  • MUC1 cytoplasmic tail
  • NMR
  • P-LL37

ASJC Scopus subject areas

  • Biotechnology

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