SELECTIVE EXTRACTION OF BACTERIAL MACROMOLECULES BY TEMPERATURE-INDUCED PHASE-SEPARATION IN TRITON X-114 SOLUTION

A Triton X-114-based extraction and temperature-dependent two phase se paration procedure was applied to a range of bacterial species of inte rest in veterinary medicine, in order to examine its usefulness for th e extraction and purification of bacterial components of value in diag nostic or vaccine applications. The bacteria were a rough gram-negativ e species (Brucella ovis), three smooth gram-negative species (Yersini a enterocolitica, Brucella abortus and Bordetella bronchiseptica), a g ram-positive species (Colynebacterium pseudotuberculosis), and a mycob acterium (M. paratuberculosis). Macromolecules were characterised by S DS-polyacrylamide gel electrophoresis by using Coomassie blue and silv er 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. A fter separation about 30 proteins were found in the aqueous phase, whi le the detergent phase contained predominantly rough lipopolysaccharid e and two proteins of about 29 kDa. After separation by preparative SD S-polyacrylamide gel electrophoresis and with the aid of monoclonal an tibodies 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 specie s varied, ranging from smooth lipopolysaccharides and proteins in case of the gram-negative bacteria, to mostly polysaccharides from the myc obacterium and mostly proteins from the gram-positive bacterium. Inter estingly, components which were identified to be of importance in the antibody response during infections, were predominantly found in the d etergent phases of all examined bacterial extracts.