Expression and purification of a recombinant LL-37 from Escherichia coli

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Human cathelicidin-derived LL-37 is a 37-residue cationic, amphipathic α-helical peptide. It is an active component of mammalian innate immunity. LL-37 has several biological functions including a broad spectrum of antimicrobial activities and LPS-neutralizing activity. In order to determine the high-resolution three-dimensional structure of LL-37 using NMR spectroscopy, it is important to obtain the peptide with isotopic labels such as 15N, 13C and/or 2H. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify LL-37 using Glutathione S-transferase (GST) fusion system. LL-37 gene was inserted into vector pGEX-4T3 and expressed as a GST-LL-37 fusion protein in BL21(DE3) strain. The recombinant GST-LL-37 protein was purified with a yield of 8 mg/l by affinity chromatography and analyzed its biochemical and spectroscopic properties. Factor Xa was used to cleave a 4.5-kDa LL-37 from the GST-LL-37 fusion protein and the peptide was purified using a reverse-phase HPLC on a Vydac C18 column with a final yield of 0.3 mg/l. The protein purified using reverse-phase HPLC was confirmed to be LL-37 by the analyses of Western blot and MALDI-TOF-Mass spectrometry. E. coli cells harboring the expression vector pGEX-4T3-LL-37 were grown in the presence of the 15N-labeled M9 minimal medium and culture conditions were optimized to obtain uniform 15N enrichment in the constitutively expressed LL-37 peptide. These results suggest that our production method will be useful in obtaining a large quantity of recombinant LL-37 peptide for NMR studies. © 2006 Elsevier B.V. All rights reserved.




Moon, J. Y., Henzler-Wildman, K. A., & Ramamoorthy, A. (2006). Expression and purification of a recombinant LL-37 from Escherichia coli. Biochimica et Biophysica Acta - Biomembranes, 1758(9), 1351–1358.

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