title: Simple method for purification of enterotoxigenic Escherichia coli fimbriae.
abstract: Enterotoxigenic Escherichia coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera.
authors.affiliation: Center for Vaccine Development, University of Maryland Medical School, Baltimore, MD, USA; Department of Medicine, University of Maryland Medical School, Baltimore, MD, USA.
authors.forename: Brittany
authors.initials: B
authors.lastname: Curtis
authors.affiliation: Center for Vaccine Development, University of Maryland Medical School, Baltimore, MD, USA; Department of Medicine, University of Maryland Medical School, Baltimore, MD, USA.
authors.forename: Christen
authors.initials: C
authors.lastname: Grassel
authors.affiliation: Center for Vaccine Development, University of Maryland Medical School, Baltimore, MD, USA; Department of Medicine, University of Maryland Medical School, Baltimore, MD, USA.
authors.forename: Rachel S
authors.initials: RS
authors.lastname: Laufer
authors.affiliation: Center for Vaccine Development, University of Maryland Medical School, Baltimore, MD, USA; Department of Pediatrics, University of Maryland Medical School, Baltimore, MD, USA.
authors.forename: Khandra T
authors.initials: KT
authors.lastname: Sears
authors.affiliation: Center for Vaccine Development, University of Maryland Medical School, Baltimore, MD, USA; Department of Pediatrics, University of Maryland Medical School, Baltimore, MD, USA.
authors.forename: Marcela F
authors.initials: MF
authors.lastname: Pasetti
authors.affiliation: Center for Vaccine Development, University of Maryland Medical School, Baltimore, MD, USA; Department of Medicine, University of Maryland Medical School, Baltimore, MD, USA.
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Barry
authors.affiliation: Center for Vaccine Development, University of Maryland Medical School, Baltimore, MD, USA; Department of Medicine, University of Maryland Medical School, Baltimore, MD, USA. Electronic address: rsimon@medicine.umaryland.edu.
authors.forename: Raphael
authors.initials: R
authors.lastname: Simon
mesh_terms: D000907:Antibodies, Bacterial / Q000097:blood; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D054307:Enterotoxigenic Escherichia coli / Q000276:immunology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D022361:Escherichia coli Vaccines / Q000276:immunology; D036781:Fimbriae Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D010861:Fimbriae, Bacterial; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D014461:Ultracentrifugation
chemical_list: D000907:Antibodies, Bacterial; D029968:Escherichia coli Proteins; D022361:Escherichia coli Vaccines; D007074:Immunoglobulin G; D036781:Fimbriae Proteins
keywords: Escherichia coli; Fimbriae; Purification