Identification of proteins that interact with podocin using the yeast 2-hybrid system.
OBJECTIVE
As a membrane protein at the insertion site of the slit diaphragm (SD) complex in podocyte foot processes, podocin has been reported to act as a scaffolding protein required to maintain or regulate the structural integrity of the SD. In order to identify proteins that associate or interact with podocin, we screened a mouse kidney complementary DNA (cDNA) library using a yeast 2-hybrid system.


METHODS
1) The full-length cDNA of podocin from the mouse kidney was amplified by Polymerase Chain Reaction (PCR), 2) The PCR product was cloned into a pGBKT7 vector, pGBKT7-podocin, 3) After the pGBKT7-podocin was transformed into AH109, the AH109/pGBKT7-podocin product was obtained, 4) The mouse kidney cDNA library was transformed into the AH109/pGBKT7-podocin and screened by selection steps, 5) Next, twelve clones were cultured and isolated, 6) The yeast-purified plasmids were transformed into Escherichia coli (E. coli) by heat shock, and 7) To identify the activation domain (AD)/library inserts, we digested them with Him III, and the fragments were then sequenced.


RESULTS
12 positive clones that interacted with podocin were obtained by screening a mouse kidney cDNA library using pGBKT7-podocin. Among them, only 4 clones were found to function at the podocyte where podocin is present.


CONCLUSIONS
Additional studies are needed to clarify the role and interaction with podocin and candidates.
Park
Soo Jin
SJ
Clinical Research Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Gangnam-Gu, Seoul, Korea.
Lee
Byung Ha
BH
Kim
Dae Joong
DJ
D000818:Animals; D003001:Cloning, Molecular; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020798:Two-Hybrid System Techniques
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C407223:NPHS2 protein