title: Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis.
abstract: Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translationally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria.
authors.affiliation: Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, St. Lucia, Qld. 4072, Australia.
authors.forename: Manuela
authors.initials: M
authors.lastname: Dieckelmann
authors.forename: Louise F
authors.initials: LF
authors.lastname: Roddam
authors.forename: Michael P
authors.initials: MP
authors.lastname: Jennings
mesh_terms: D015153:Blotting, Western; D036781:Fimbriae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D006639:Histidine / Q000378:metabolism*; D015394:Molecular Structure; D009345:Neisseria meningitidis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D036781:Fimbriae Proteins; D006639:Histidine