B-880. Direct Interaction Between a Serine/Threonine Kinase PknA and a Putative Cell Division Protein Wag31 in Mycobacterium tuberculosis
Session: Slide Session: Mycobacteria: New Therapies, Mechanisms and Models
Sunday, October 26, 2008: 12:00 AM
Room: Independence I (Grand Hyatt)
Background: PknA is a serine/threonine kinase (eukaryotic-like protein kinase) in Mycobacterium tuberculosis. In vitro peptide library screen showed that Wag31, a putative cell division protein, was a new substrate phospholyated by PknA. The signal transduction pathway involving Wag31 and PknA plays a unique role in M. tuberculosis growth regulation that may participate in the pathogenesis of tuberculosis. However, it is not unknown whether there is the direct interaction between PknA and Wag31. Methods: PknA and Wag31 genes were cloned from M. tuberculosis. PknA gene was expressed by pET30 system to produce the pET30/His6-PknA plasmid. His6-PknA was purified by His-Bind column and Ion-Exchange column. Wag31 gene was amplified by PCR with specific primers encoding the LIC overhang. The amplified gene was inserted into an expression plasmid pMCSG19 to produce the pMCSG19/Wag31 plasmid. Wag31 was purified by His-Bind column and Gel Filtration column. More than 1 mg of each protein (PknA or Wag31) were obtained. And then we performed Far-Western blotting. Results: Wag31, purified by His-Bind column and Gel Filtration column, showed a single protein band of 28kDa in SDS-PAGE. Purified His6-PknA was incubated with nitrocellulose membranes on which Wag31 and BSA proteins were transferred after SDS-PAGE. After removal of unbound proteins by washing, the bound His6-PknA (His6-PknA bound to Wag31) was detected by anti-His-tag antibody, showing direct protein-protein interaction. Conclusions: These results reveal that there is the direct interaction between PknA and Wag31, which identifies a novel target for the development of new anti-tuberculars (new inhibitors to the direct interaction).
Ha Sun1, Jae Lee1, Jung Lee, MS2, Sang Lee, Dr3, Seung Sohn1 and  S. H. Lee, None., (1)Myongji University, (2)Asan Medical Center, Seoul, Korea, Republic of, (3)Myongji University, Yongin, Korea, Republic of

Current Licenses and Certification
Korean License of Medical Doctor
Education
Mar, 1996 - Feb, 2002
University of Ulsan, College of Medicine, Seoul, Korea
Professional Experiences
Internship
Mar, 2002- Feb, 2003
Asan Medical Center, Seoul, Korea
Residency
Mar, 2004-
Department of Internal Medicine,
Asan Medical Center, Seoul, Korea