Identification of proteins that interact with podocin using the yeast 2-hybrid system

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.

Keywords: Podocyte; mouse kidney complementary DNA library; podocin; yeast 2-hybrid system.

Fig. 1

Confirmation of the recombinant bait expression by restriction enzyme (REs), EcoR I and BamH I, and digestion. The podocin and vector that had been transformed to DH5α appeared on the LB plate. After grown in the LB medium, the resulting plasmid was divided into 2 bands, podocin and vector, by the REs. (Marker: 0.25-10 KB DNA ladder marker.)

Fig. 2

Colony PCR from the pGBKT7-podocin transformed into the AH109 by the lithium acetate-mediated method. By cPCR using a podocin primer, the colonies on the SD/-Ura plate were confirmed to be podocin. PCR, polymerase chain reaction; cPCR, colony polymerase chain reaction.

Fig. 3

Mouse kidney cDNA library that was transformed into AH109/pGBKT7-podocin. (A) Clones that appeared on the SD/-Trp/-Leu plate (low stringency). (B) Putative clones that were transferred to the SD/-Trp/-Leu/-His plate (medium stringency). The copy number of independent clones in the library was screened at least 1.5-3 times at this step. CDNA, complementary DNA.

Fig. 4

The putative proteins predicted to interact with podocin. (A) On the SD/-Trp/-Leu/-His/X-αgal plate, the blue clones were the AD/library plasmids mated with podocin. (B) Without X-α-gal.

Fig. 5

Rescue AD/Library plasmids via transformation of E. coli. The yeastpurified plasmid DNA was transformed into E. coli and plated on LB medium containing ampicillin. Then, the plasmids were digested with Hind III and resulted in various patterns such as (A), (B), (C), and (D).