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 (15)N 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 (), 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.
authors.affiliation: The Structure-Fun Laboratory, Eppley Institute for Research in Cancer and Allied Diseases, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, 68198-6805, USA.
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Li
authors.forename: Xia
authors.initials: X
authors.lastname: Li
authors.forename: Guangshun
authors.initials: G
authors.lastname: Wang
mesh_terms: D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007553:Isotope Labeling; D018396:Mucin-1; D009077:Mucins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D054804:Cathelicidins
chemical_list: D000951:Antigens, Neoplasm; D023181:Antimicrobial Cationic Peptides; C060192:MUC1 protein, human; D018396:Mucin-1; D009077:Mucins; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D054804:Cathelicidins