Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli.
Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for (15)N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.
Laboratoire Ecologie et Biologie des Interactions UMR CNRS 7267, Equipe Microbiologie de l'Eau, Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France. julien.verdon@univ-poitiers.fr
Julien
J
Verdon
Nicolas
N
Girardin
Adrienne
A
Marchand
Yann
Y
Héchard
Jean-Marc
JM
Berjeaud
D001430:Bacteriocins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007875:Legionella / Q000187:drug effects*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D001430:Bacteriocins; D011994:Recombinant Proteins; C558932:warnericin RK, Staphylococcus warneri