Identification of Proteins That Interact with Podocin Using the Yeast 2-Hybrid System.
10.3349/ymj.2009.50.2.273
- Author:
Soo Jin PARK
1
;
Byung Ha LEE
;
Dae Joong KIM
Author Information
1. Clinical Research Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea.
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
Podocyte;
podocin;
mouse kidney complementary DNA library;
yeast 2-hybrid system
- MeSH:
Animals;
Cloning, Molecular;
Intracellular Signaling Peptides and Proteins/genetics/*metabolism;
Membrane Proteins/genetics/*metabolism;
Mice;
Polymerase Chain Reaction;
Protein Binding;
*Two-Hybrid System Techniques
- From:Yonsei Medical Journal
2009;50(2):273-279
- CountryRepublic of Korea
- Language:English
-
Abstract:
PURPOSE: 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. MATERIALS AND 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. CONCLUSION: Additional studies are needed to clarify the role and interaction with podocin and candidates.
