Expression and purification of native and truncated forms of CadF, an outer membrane protein of Campylobacter.
Campylobacter is now recognized as the most common bacterial agent of gastroenteritis. The adhesion of bacteria to intestinal cells is a major step in human colonization. The binding of Campylobacter jejuni cells to fibronectin (Fn), a component of the extra cellular matrix, is mediated by a 37,000 outer membrane protein termed CadF for Campylobacter adhesion to Fn. CadF protein is very hard to purify from Campylobacter membranes. In order to study the conformation of this protein, we set out to clone, express, purify, and re-fold the CadF protein. The nucleotide sequence encoding the N-terminal domain of the CadF protein was cloned in a pET-based expression vector. The recombinant protein was further produced in Escherichia coli, purified from inclusion bodies, and refolded. More specifically, the purification experiments were set-up as follows: (i) protein aggregates were collected from cell-lysates, solubilized in urea and enriched by ion-exchange chromatography; (ii) refolding was achieved by drop-by-drop dilution method in detergent containing buffer and monitored by CD measurements; (iii) the protein was finally purified to homogeneity by gel filtration chromatography. In spite of our success in purifying the N-terminal domain of the CadF protein, repeated attempts to express and purify the entire cadF gene in E. coli failed. Using a novel approach, we found it possible to express the entire cadF gene fused to a hexa-histidine encoding nucleotide sequence in C. jejuni. This allowed the expression, synthesis, and purification of the recombinant CadF-His tagged protein from C. jejuni by nickel affinity chromatography followed by gel filtration chromatography. In summary, we developed a novel strategy to produce significant quantities of a recombinant N-terminal portion of the CadF protein (46.5 microg/mg of bacterial dry weight) and of the native CadF protein (3.5 microg/mg of bacterial dry weight) for further studies.
Mamelli
Laurent
L
EA 2197, IFR 48, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille Cedex 05, France.
Pagès
Jean-Marie
JM
Konkel
Michael E
ME
Bolla
Jean-Michel
JM
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D016123:Campylobacter jejuni / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D001425:Bacterial Outer Membrane Proteins; C107872:CadF protein, Campylobacter jejuni; D002352:Carrier Proteins; D011994:Recombinant Proteins