High resolution electroelution of polyacrylamide gels for the purification of single proteins from Mycobacterium tuberculosis culture filtrate

Abstract

Culture filtrate from Mycobacterium tuberculosis contains protective molecules which have been used successfully in experimental vaccines against tuberculosis. Despite an increasing number of mycobacterial proteins being characterised, a major effort is still needed to get an overview of the many potentially interesting molecules in culture filtrate. In this study we describe a high throughput method for purification and biological evaluation of protein components in complex protein mixtures. The method presents a new application of the recently developed Mini Whole Gel Eluter and employs this apparatus for the high resolution electroelution of selected molecular mass fractions of protein mixtures previously separated in large polyacrylamide gels. Two novel M. tuberculosis culture filtrate proteins (CspA and TB18.6) were purified by this method, their N-terminal sequences were determined and the open reading frame encoding each of the proteins identified. The immunological recognition of the molecules were evaluated in tuberculosis infected mice and guinea pigs. Both proteins induced DTH responses in guinea pigs and IFN-gamma release from spleen lymphocytes isolated from infected mice.