Journal: Microbiology Spectrum

Article Title: Mycobacterium tuberculosis Rv1324 Protein Contributes to Mycobacterial Persistence and Causes Pathological Lung Injury in Mice by Inducing Ferroptosis

doi: 10.1128/spectrum.02526-22

Figure Lengend Snippet: Expression and activity of the recombinant Rv1324 protein. (A and B) The expression of the Rv1324 protein in the Rv1324/Msm strain was analyzed by SDS-PAGE (A) and Western blotting (B). The supernatant of the Vec/Msm and Rv1324/Msm strains were collected. 20 micrograms of the whole-cell lysate proteins of Rv1324/Msm strains were added to the SDS-PAGE gels to analyze the Rv1324 protein expression. For the Western blotting, the proteins bands were visualized in NBT/BCIP solution. The whole-cell lysate proteins of Vec/Msm were used as a control protein. Lane 1 represents the proteins expressed in the supernatant of the Vec/Msm strain. Lane 2 represents the protein expressed in the supernatant of Rv1324/Msm strain number 1. Lane M represents the PageRuler prestained protein ladder. (C) The subcellular localization of the recombinant Rv1324 protein in the Rv1324/Msm strain detected by Western blotting. The proteins bands were visualized using WesternBright ECL detection reagents. WCL represents the whole-cell lysate proteins. CW represents the cell wall fraction. CM represents the cell membrane fraction. SOL represents the soluble fraction of the Rv1324/Msm cells. The expression of the GroEL protein was used as a control protein. (D and E) Analysis of Rv1324 protein expression by SDS-PAGE (D) and Western blotting (E). For the Western blotting, the proteins bands were visualized in NBT/BCIP solution. Lane 1 represents the proteins expressed in the supernatant of E. coli BL21(DE3). Lanes 2 to 4 represent the protein expressed in the supernatant of pCold-Rv1324/ E. coli BL21(DE3) strains numbers 1 to 3. Lane M represents the PageRuler prestained protein ladder. (F) SDS-PAGE analysis of the purified recombinant Rv1324 and Rv1324m proteins. (G) Assessment of the antioxidant activity of the recombinant Rv1324 and TrxC proteins. M. tuberculosis TrxC, which was used as a control protein, was also constructed, expressed, and purified using the same protocol with Rv1324. (H) Assessment of the antioxidant activity of the recombinant Rv1324 and Rv1324m proteins.

Article Snippet: Finally, the protein bands were visualized using WesternBright ECL detection reagents (Advansta, China).

Techniques: Expressing, Activity Assay, Recombinant, SDS Page, Western Blot, Purification, Antioxidant Activity Assay, Construct