Separation and Fractionation of Cell Wall and Cell Membrane Proteins from <em>Mycobacterium tuberculosis</em> for Downstream Protein Analysis

Mycobacterium cell wall and membrane proteins, which play a central role in tuberculosis pathogenesis, were successfully separated using preparative Isoelectric Focusing (IEF) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Followed by gel elution, overcoming limitations in conventional methods for the separation of hydrophobic proteins. In this procedure, M. tuberculosis colonies were transferred from Lowenstein-Jensen slants into 2 mL of 7H9 broth, dispersed with glass beads, and incubated at 37 °C for 2 weeks. Then, the culture was scaled up to 200 mL and grown in a shaker for 4 weeks. It was further upscaled to 1 L with 500 mL of 7H9 broth and grown for an additional 4 weeks. Grown Mycobacteria were pelleted by centrifugation at 1741 × g for 30 min. For each 2 g pellet, 1 mL of breaking buffer was added, and the sample was sonicated. The lysate was centrifuged at 3436 × g for 15 min to remove unbroken cells, and the supernatant was concentrated. This supernatant (whole cell lysate) was centrifuged at 13751 × g for 30 min to pellet cell wall proteins. The remaining supernatant was ultra-centrifuged at 100,000 × g for 4 h to separate the cell membrane and cytosol. The isolated cell wall and membrane proteins were loaded onto a liquid preparative IEF system at 4 °C and separated at 12 W until the voltage stabilized at 1400 V, which separates 20 fractions. These IEF fractions were further separated by preparative SDS-PAGE, and proteins were eluted using a whole gel eluter at 250 mA, resulting in 30 fractions. Through this protocol, we were able to identify novel M. tuberculosis cell walls and membrane-specific biomarkers, and it also shows potential for characterizing similar proteins in other pathogens.