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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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Li Y,Li X,Wang G

doi

10.1016/j.pep.2005.10.022

keywords:

subject

Has Abstract

pub_date

2006-06-01 00:00:00

pages

498-505

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(05)00380-3

journal_volume

47

pub_type

杂志文章

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