Screening and Selection of Proteins Interacting with Myocilin.

Cho, Myoung Kuk; Son, Sung Su; Kee, Chang Won

Original Article

Journal of the Korean Ophthalmological Society 2006;47(4):637-642.
Published online April 30, 2006.

Screening and Selection of Proteins Interacting with Myocilin.

Myoung Kuk Cho, Sung Su Son, Chang Won Kee

Department of Ophthalmology, Samsung Medical Center, College of Medicine, Sungkyunkwan University, Seoul, Korea. cwkee@smc.samsung.co.kr

마이오실린과 상호작용하는 단백질 탐색 및 선별

조명국,손성수,기창원

Department of Ophthalmology, Samsung Medical Center, College of Medicine, Sungkyunkwan University, Seoul, Korea

Correspondence: Chang Won Kee, M.D.

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.

Key Words: Cytoskeleton;Myocilin;Yeast two-hybrid;Interaction;Trabecular meshwork cell