Isolation and Purification of low molecular weight proteins from culture filtrate of Mycobacterium tuberculosis Strain C (ATCC 35808)

Document Type: Original Articles

Authors

1 Department of Biological Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran

2 Department of Tuberculin and Mallein Production & Research, Razi Vaccine and Serum Research Institute, Alborz, Karaj, Iran

3 Department of Microbiology, Arak University of Medical Sciences, Arak, Iran

Abstract

In last couple of years a number of new and rapid tests for the diagnosis of Tuberculosis based on the low molecular weight antigens from Mycobacterium tuberculosis (Mtb) culture supernatant have been developed. In this study we aimed to isolate and purify low molecular weight antigens that are secreted by Mtb strain C, for diagnostic purpose. The secretory proteins from culture filtrate of Mtb were extracted using Ammonium sulphate precipitations and Sephadex G50 gel chromatography. The obtained antigen fractions were analyzed for their protein concentrations and approximate molecular weight by Lowry method and SDS - PAGE (12.5%), respectively. DOT - ELISA and Western Blot assay using sera from pulmonary tuberculosis patients (polyclonal antibodies) was performed to confirm the presence of purified low molecular weight proteins isolated from Mtb. During chromatography, low molecular weight proteins were separated, that was approximately 0.7 mg/ml of the total proteins (1.662 mg/ml). During SDS - PAGE analysis, the purified protein fractions appeared with molecular weights in the range of 14 kDa to 41 kDa. By Western Blotting the chromatographic fraction using tuberculosis patients’ sera we were able to identify bands in the range of 30 kDa to 41 kDa. The low molecular weight proteins present in the culture filtrate of Mtb strain C were purified by ammonium sulphate and chromatography. These fractions were confirmed by western blotting. The obtained results might support the hypothesis that the Mtb culture filtrate antigens could be used as a rapid and sensitive assay for detection of patients with pulmonary TB.

Keywords