Expression, purification, and refolding of biologically active Acinetobacter baumannii OmpA from Escherichia coli inclusion bodies.
Infections caused by Acinetobacter baumannii have emerged as a significant clinical problem due to the increase in infections caused by antibiotic resistant strains. A. baumannii OmpA is a highly conserved membrane protein that has multiple roles in interacting with the host during infection, and thus represents an attractive target for the development of novel antibacterial therapies. In the present study, the coding sequence of the mature form of A. baumannii OmpA was cloned into the vector pET-15b and purified under denaturing conditions from Escherichia coli inclusion bodies using nickel affinity chromatography. A Triton X-114 wash step was incorporated into the purification method in order to remove endotoxin, resulting in endotoxin levels of <1.3 EU/mg of protein. A protocol was developed for refolding the purified protein by dilution into the non-ionic detergent n-octyl-β-D-glucopyranoside followed by dialysis to remove excess denaturant and detergent. Cytotoxicity assays demonstrated that refolded A. baumannii OmpA was able to induce cell death in A549 cells. In addition, a polyclonal antibody was raised against the refolded protein and used to assess extracellular secretion of OmpA by Western blot. This protein expression and purification system may be useful for further characterization of A. baumannii OmpA.
Unit of Infectious Disease, Microbiology, and Preventive Medicine, Institute of Biomedicine of Sevilla, University Hospital Virgen del Rocío/CSIC/University of Sevilla, 41013 Sevilla, Spain. mcconnell.mike75@gmail.com
Michael J
MJ
McConnell
Jerónimo
J
Pachón
D040981:Acinetobacter baumannii / Q000235:genetics* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D045744:Cell Line, Tumor; D002470:Cell Survival; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; C078167:OMPA outer membrane proteins