Improved procedure for the isolation and purification of lipoarabinomannan from Mycobacterium bovis strain AN5

The effectiveness of a Triton X-114 phase separation technique for the isolation of lipoarabinomannan from mycobacteria was investigated. Lipophilic compounds were extracted from disrupted Mycobacterium bovis AN5 by a temperature-induced phase separation in aqueous Triton X-114 solution. In combination with nuclease and protease digestion and subsequent purification by ultracentrifugation, this isolation procedure provided mycobacterial lipoglycans that were devoid of contaminating protein or nucleic acid. The purity of the extract was monitored throughout the isolation procedure by physicochemical techniques, including, electrophoresis, gas–liquid and liquid chromatography and microanalysis. Chromatographic fractionation of the purified extract by gel size exclusion chromatography, performed with deoxycholate buffer, resulted in the isolation of different size classes of lipopolysaccharide, corresponding to lipoarabinomannan, lipomannan and phosphatidylinositolmannan. This novel application of Triton X-114 phase partitioning provided a fast, mild and efficient method for the isolation of highly purified mycobacterial lipoglycan.