Availability of genome sequence of Mycobacterium tuberculosis has accelerated identification of antigens for serodiagnosis of tuberculosis and a number of new antigens are being tested in various combinations to produce cocktails with high sensitivity and specificity. For producing a highly specific diagnostic test, it is important that the recombinant antigens be highly pure, free of host protein, and correctly folded so that they bind only to specific antibodies. Also, for commercial viability they need to be produced in high yields. We have cloned, expressed, and purified a number of mycobacterial antigens in Escherichia coli. This paper describes, expression and purification of two important mycobacterial proteins with serodiagnostic potential, namely, 38-kDa and Mtb81 antigens, in monomeric form. The protocol involves using a T7 promoter based expression vector under conditions of regulated and slow expression followed by three-step column chromatography procedure to obtain highly purified proteins. The yields of the two proteins were several folds higher than previously reported. The purified proteins were useful in detecting antibodies in sera of tuberculosis patients (smear positive, smear negative, and extra-pulmonary categories). (c) 2004 Elsevier Inc. All rights reserved.