J Korean Ophthalmol Soc.  2006 Apr;47(4):637-642.

Screening and Selection of Proteins Interacting with Myocilin

• Cho MK
• Son SS
• Kee CW

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.

Keyword

Cytoskeleton; Myocilin; Yeast two-hybrid; Interaction; Trabecular meshwork cell

MeSH Terms

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
Actins
Carrier Proteins
Myosin Light Chains
Myosins