Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins.
PURPOSE: To maximize the expression level of myocilin and its truncated proteins in Escherichia coli (E. coli) and to examine the biological effects of bacterially expressed myocilin as compared to eukaryotic myocilin on cultured human trabecular meshwork (TM) cells. METHODS: Myocilin full length (1-504 amino acids) and two truncated proteins, myocilin 1-270 and 271-504, were expressed and purified from an E. coli strain, Rosetta2(DE3)pLysS. The eukaryotic myocilin was purified from cultured medium of a transformed TM cell line (TM5) transduced with feline immunodeficiency virus that contains an internal cassette expressing full length myocilin. The morphology and adhesion of human TM cells plated either on fibronectin alone or on fibronectin/purified myocilin mixtures were assessed by phase contrast microscopy. Actin cytoskeleton was examined using Oregon Green phalloidin. Immunofluorescence staining for paxillin was also performed. RESULTS: The expression of full length and truncated myocilin proteins in Rosetta2(DE3)pLysS was markedly increased especially when the bacteria were grown in media supplemented with 1.0% glucose. Cell adhesion was impaired and microspikes were formed when TM cells were plated onto fibronectin/bacterial full length myocilin mixtures. Loss of actin stress fibers and focal adhesions was also observed. This myocilin phenotype was also seen with myocilin 1-270, but not with myocilin 271-504. The eukaryotic full length myocilin produced nearly identical de-adhesive effects as those of the bacterially expressed myocilin. CONCLUSIONS: The condition for a high level expression of full length and truncated myocilins in E. coli was optimized. The bacterial and eukaryotic recombinant full length myocilin produced similar biological consequence on TM cells. The myocilin phenotype appears to be largely due to the NH(2)-terminal half of the protein.[1]References
- Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins. Park, B.C., Shen, X., Fautsch, M.P., Tibudan, M., Johnson, D.H., Yue, B.Y. Mol. Vis. (2006) [Pubmed]
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