Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

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-translation ally 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 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x 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. (C) 2003 Elsevier Science (USA). All rights reserved.

The synthesis and X-ray crystal structure of 6-bromo-2,4,4-trimethyl-cyclohex-2-enone

Regiospecific bromination of 2,4,4-trimethyl-cyclohex-2-enone was achieved and the X-ray crystal structure of 6-bromo-2,4,4-trimethyl-cyclohex-2-enone is presented.

Cherbourg revisited: hearing health changes in an Aboriginal community, 1972 to 2000

The aim of this study was to investigate current audiological outcomes of Cherbourg schoolchildren and compare these outcomes to those obtained in a 1972 study of hearing health in the same community. Seventy-eight primary school children of Cherbourg State School participated in the study. Their peripheral hearing and middle ear function were tested using pure-tone air conduction audiometry and tympanometry respectively. A significant improvement in the hearing status of this population was noted compared to that reported in 1972. The improvements in the hearing status of Indigenous schoolchildren at Cherbourg can be attributed to a number of factors, including increased awareness of both ear health and general health, as well as the introduction of hearing health care services, over the past three decades.