orals 5th Asia-Pacific NMR Symposium 2013

High-yield expression and NMR structural studies of Antimicrobial Peptides  (#60)

Yongae KIM 1
  1. Department of Chemistry and Protein Research Center for Bio-Industry, Hankuk University of Foreign Stuides, Yong-In, 449-791, Korea

Drugs isolated from organic compounds are a long established part of our defense against bacterial pathogens. Their wide use has led to pathogens' increased drug resistance and the need to find novel classes of antimicrobial peptides as alternatives to antibiotics. Lactophoricin (LPcin), a cationic amphipathic peptide consists of 23-mer peptide, corresponds to the carboxy terminal 113–135 region of component-3 of proteose-peptone. LPcin is a good candidate as a peptide antibiotic because it has an antibacterial activity but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 which has mutant amino acids, LPcin-yk1 and LPcin-yk3 that has shorter mutant amino acids are recently developed by using peptide engineering in our laboratory. These three LPcin analogs show better antibioitic activities than wild-type LPcin and no toxicity at all.

In order to understand the structural correlation between LPcin analogs structure and antimicrobial activity under the membrane environments, we tried to express and purify as large as amounts of LPcin and three different LPcin analogs. We finally optimized and succeed to overexpress in the form of fusion protein in Escherichia coli and purified with biophysical techniques like Ni-affinity chromatography, dialysis, centrifuge, chemical cleavage, and reversed-phase semiprep HPLC. In here, we will present the optimizing processes for high-yield expression and purification and solution NMR spectra and solid state NMR spectra for antimicrobial mechanisms.

  1. Tae-Joon Park, Sung-Sub Choi, Ji-Sun Kim, and Yongae Kim (2008) “Expression, Purification, and NMR structural studies of antibacterial peptide, bovine lactophoricin” The 1st Italy-Korea Symposium on Antimicrobial Peptides Proceedings 1, P14
  2. Tae-Joon Park, Ji-Sun Kim, Sung-Sub Choi, and Yongae Kim (2009) “Cloning, Expression, Isotope Labeling, Purification, and Characterization of Bovine Antimicrobial Peptide, Lactophoricin in E. Coli” Prot. Exp. Purification 65, 1, 23
  3. Tae-Joon Park, Ji-Sun Kim, Hee-Chul Ahn, and Yongae Kim (2011) “Solution and Solid-state NMR Structural Studies of Antimicrobial Peptides, LPcin-I and LPcin-II.” Biophy. J. 101, 5, 1193