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 journalArticle

58 Citations (Scopus)

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 1 2006

Fingerprint

Isotope Labeling
Cloning
Isotopes
Labeling
Escherichia coli
Purification
Organism Cloning
formic acid
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Peptides
Fusion reactions
Affinity chromatography
Cyanogen Bromide
Thioredoxins
Proteins
Dipeptides
DNA sequences
Electrophoresis
Affinity Chromatography

Keywords

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

ASJC Scopus subject areas

  • Biotechnology

Cite this

Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies. / Li, Yifeng; Li, Xia; Wang, Guangshun.

In: Protein Expression and Purification, Vol. 47, No. 2, 01.06.2006, p. 498-505.

Research output: Contribution to journalArticle

@article{f21c8f616b674eac90b6aba7f834b86e,
title = "Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies",
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.",
keywords = "Antimicrobial peptide, Chemical cleavage, Escherichia coli, Formic acid, Isotope labeling, LL-37, MUC1 cytoplasmic tail, NMR, P-LL37",
author = "Yifeng Li and Xia Li and Guangshun Wang",
year = "2006",
month = "6",
day = "1",
doi = "10.1016/j.pep.2005.10.022",
language = "English (US)",
volume = "47",
pages = "498--505",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies

AU - Li, Yifeng

AU - Li, Xia

AU - Wang, Guangshun

PY - 2006/6/1

Y1 - 2006/6/1

N2 - 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.

AB - 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.

KW - Antimicrobial peptide

KW - Chemical cleavage

KW - Escherichia coli

KW - Formic acid

KW - Isotope labeling

KW - LL-37

KW - MUC1 cytoplasmic tail

KW - NMR

KW - P-LL37

UR - http://www.scopus.com/inward/record.url?scp=33646726320&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33646726320&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2005.10.022

DO - 10.1016/j.pep.2005.10.022

M3 - Article

VL - 47

SP - 498

EP - 505

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -