Identification of PITX2 interacting proteins using both protein affinity and a novel human trabecular meshwork yeast two hybrid cDNA library

Abstract

Abstract: : Purpose:Mutations in the transcription factor PITX2 cause Axenfeld–Rieger malformations often leading to glaucoma. We hypothesize that for proper function, PITX2 interacts with other proteins and that mutations in PITX2 can affect these protein–protein interactions. Altered PITX2 protein–protein interactions may be key factors leading to the disease phenotype. Methods:To identify PITX2 interacting proteins, we are using both a protein affinity and a yeast two–hybrid approach. PITX2 was expressed in bacteria and purified using nickel–agarose beads. Approximately 5000 mg of ocular protein lysate were extracted from 150 porcine eyes. The porcine ocular protein extract and the recombinant PITX2 are being used in pull–down experiments to isolate PITX2 interacting proteins. Columns with no recombinant protein and with bacterially expressed LACZ are used as negative controls. In parallel, poly–A RNA was isolated from a human trabecular meshwork (TM) primary cell line. This RNA was used to create a TM yeast two–hybrid cDNA library that incorporates Gateway recombination technology (Invitrogen). RT–PCR was initially performed on the cell line total RNA to confirm PITX2 expression indicating that this cDNA library is an excellent reagent to isolate PITX2 interacting proteins. Results: We have successfully expressed and purified recombinant PITX2. A human trabecular meshwork primary cell line cDNA library has been created in the pEXP–AD 502 vector. The amplified library contains 7.8x10e9 cfu (2.1x10e6 primary cfu) with 96% of colonies containing inserts with an average insert size of 1.9 kb. Conclusions: Porcine eyes appear to be an excellent source of protein extracts to isolate factors interacting with PITX2. The novel TM yeast two–hybrid cDNA library is an excellent and unique tool to identify genes involved in ocular function. Two–hybrid analyses of the cDNA library using known glaucoma genes as bait are in progress. Identification of PITX2 interacting proteins will lead to a further understanding of PITX2 cellular function as well as glaucoma pathogenesis. Identified proteins/genes will also serve as candidates for other eye phenotypes.

Keywords: anterior segment • transcription factors • protein purification and characterization