Screening and Selection of Proteins Interacting with Myocilin.
- Author:
Myoung Kuk CHO
1
;
Sung Su SON
;
Chang Won KEE
Author Information
1. Department of Ophthalmology, Samsung Medical Center, College of Medicine, Sungkyunkwan University, Seoul, Korea. cwkee@smc.samsung.co.kr
- Publication Type:Original Article
- Keywords:
Cytoskeleton;
Myocilin;
Yeast two-hybrid;
Interaction;
Trabecular meshwork cell
- MeSH:
Actins;
Blotting, Western;
Carrier Proteins;
Clone Cells;
Cytoskeleton;
Databases, Nucleic Acid;
Diploidy;
Gene Library;
Humans;
Mass Screening*;
Microtubules;
Muscle, Skeletal;
Myosin Light Chains;
Myosins;
Patient Dropouts;
Plasmids;
Trabecular Meshwork;
Yeasts
- From:Journal of the Korean Ophthalmological Society
2006;47(4):637-642
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
PURPOSE: To identify novel proteins which bind to myocilin, using a yeast two-hybrid screening system. METHODS: A bait plasmid of myocilin was constructed, and then transformed into yeast AH109. The transformed yeast cells were mated with yeast Y187 containing human skeletal muscle cDNA library plasmids in 2 X YPD medium. Mated diploid yeasts were plated on synthetic dropout medium (SD/-Trp-Leu-His and SD/-Trp-Leu-His-Ade). The positive clones grown on the selective medium were amplified, sequenced, and analyzed using the database of GenBank. The expression of selected genes in trabecular meshwork (TM) cells was detected by RT-PCR. RESULTS: The expression of bait protein in yeast was confirmed by Western blot analysis. Extensive screening of a cDNA library led to the selection of twenty-four positive clones which represent eight genes, including six of cytoskeleton associated proteins such as alpha-actin, myosin regulatory light chain 2A, dynactin, syntrophin alpha1, microtubule associated protein 1B, and myosin binding protein C. CONCLUSIONS: Myocilin may interact with cytoskeleton associated proteins in TM cells. Further studies on their interactions will provide functional clues of myocilin.
