Abstract：Ribosomes of pathogenic bacteria are major targets for antibiotics, and how to prepare ribosome samples with quality and quantity is a key step for structure and antibiotic studies. In particular, it is a challenge to purify enough ribosome from slow-growing Mycobacterium such as Mycobacterium tuberculosis due to slow growth rate and thick cell wall. With the optimized steps, including growing and processing bulk culture with biosafety measures, breaking the thick cell wall of Gram-positive bacteria with effective techniques, and purification methods to combine the fast protein liquid chromatography (FPLC) and sucrose gradient separation, enough ribosome samples with high purity were obtained from Mycobacterium. The prepared samples could be subjected to complex specificity studies, such as high-resolution structure studies with X-ray crystallography and cryo-electron microscopy (cryo-EM), and shed light on the mechanism of antibiotics. This improved method is applicable in other Gram-positive bacteria.
2016, Vol. 11 
