Separation and Fractionation of Culture Filtrate Proteins (CFPs) from Mycobacterium tuberculosis

Secreted proteins of Mycobacterium tuberculosis (M. tuberculosis) play a crucial role in tuberculosis pathogenesis, immune modulation, and disease progression. Understanding their composition and function is essential for identifying novel biomarkers that can aid in tuberculosis diagnosis, vaccine development, and therapeutic interventions. This study focuses on the systematic isolation, fractionation, and characterization of culture filtrate proteins (CFPs) to enable comprehensive immunological and proteomic analyses. CFPs were obtained from M. tuberculosis H37Rv cultures grown in chemically defined conditions to ensure controlled protein expression. The culture supernatant was purified through filtration and concentrated using a hollow fiber system to retain proteins above 10 kDa. Fractionation was achieved through liquid-phase isoelectric focusing, separating proteins based on their isoelectric points into 20 distinct fractions. A further resolution was performed using preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroelution, yielding 30 protein fractions. The fractionated proteins obtained through this approach provide a valuable resource for immunological profiling and mass spectrometry-based proteomic analyses. By systematically isolating and characterizing secreted proteins, this study contributes to the identification of tuberculosis-specific protein biomarkers, which could improve diagnostic accuracy and advance our understanding of M. tuberculosis pathogenesis.