Identification of Proteins That Interact with Podocin Using the Yeast 2-Hybrid System
Yonsei Medical Journal
;
: 273-279, 2009.
Artículo
en Inglés
| WPRIM
| ID: wpr-109393
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 ANDMETHODS:
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.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Unión Proteica
/
Reacción en Cadena de la Polimerasa
/
Clonación Molecular
/
Técnicas del Sistema de Dos Híbridos
/
Péptidos y Proteínas de Señalización Intracelular
/
Proteínas de la Membrana
Tipo de estudio:
Estudio diagnóstico
Límite:
Animales
Idioma:
Inglés
Revista:
Yonsei Medical Journal
Año:
2009
Tipo del documento:
Artículo
Similares
MEDLINE
...
LILACS
LIS