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Background: Protein kinase B (PknB) is critical for the survival of Mycobacterium tuberculosis (M. tuberculosis) in vitro and in hosts. It phosphorylates various enzymes involved in biosynthesis of cell wall and a particular autolysin classified as CwIM or Rv3915 has been recently identified as a PknB substrate. However, in-depth knowledge of this protein is still unknown. The aims of this study were to purify and investigate the activity of Rv3915, as well as monitor the phosphorylation of Rv3915 and the influence of phosphorylation on the activity of this protein. Results: Using the C41 E. coli strain containing a plasmid, with a gene encoding the protein, Rv3915 was either expressed alone or co-expressed with PknB. Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used to observe the amount of Rv3915 purified, anti-poly His and anti-phosphothreonine western blot techniques were used to confirm the presence and phosphorylation of Rv3915 and zymogram assays were run to examine its activity. The results showed that Rv3195 was successfully expressed and was deemed soluble when observed in soluble fraction of E. coli lysate. It was confirmed that Rv3915 is produced as a ~45kDa protein, which does not possess any muralytic activity. However a shorter version of the protein (~25kDA) was active in zymogram, suggesting that Rv3915 is activated by cleavage. Conclusion: Rv3915 was phosphorylated by PknB and phosphorylation apparently controls stability of the protein. |
