Selective extraction of bacterial macromolecules by temperature-induced phase separation in Triton X-114 solution

Abstract

A Triton X-113-based extraction and temperature-dependent two phase separation procedure was applied to a range of bacterial species of interest in veterinary medicine, in order to examine its usefulness for the extraction and purification of bacterial components of value in diagnostic or vaccine applications. The bacteria were a rough gram-negative species ( Brucella ovis), three smooth gram-negative species ( Yersinia enterocolitica, Brucella abortus and Bordetella bronchiseptica), a gram-positive species ( Corynebacterium pseudotuberculosis), and a mycobacterium ( M. paratuberculosis). Macromolecules were characterised by SDS-polyacrylamide gel electrophoresis by using Coomassie blue and silver staining combined with proteolytic digestion. Some of the extracts were further analysed in immunoblots by their reaction with sera from infected animals. Most extensively examined was the B. ovis extract. After separation about 30 proteins were found in the aqueous phase, while the detergent phase contained predominantly rough lipopolysaccharide and two proteins of about 29 kDa. After separation by preparative SDS-polyacrylamide gel electrophoresis and with the aid of monoclonal antibodies the two proteins were identified as outer membrane proteins, one of which was identical to a previously described immunodominant 29 kDa protein. Macromolecules extracted from the other bacterial species varied, ranging from smooth lipopolysaccharides and proteins in case of the gram-negative bacteria, to mostly polysaccharides from the mycobacterium and mostly proteins from the gram-positive bacterium. Interestingly, components which were identified to be of importance in the antibody response during infections, were predominantly found in the detergent phases of all examined bacterial extracts.