Optimized bacterial expression of myocilin proteins andfunctional comparison of bacterial and eukaryotic myocilins

[摘要] Purpose: To maximize the expression level of myocilin and itstruncated proteins in Escherichia coli (E. coli) and to examinethe biological effects of bacterially expressed myocilin as compared toeukaryotic myocilin on cultured human trabecular meshwork (TM) cells.Methods: Myocilin full length (1-504 amino acids) and two truncatedproteins, myocilin 1-270 and 271-504, were expressed and purified froman E. coli strain, Rosetta2(DE3)pLysS. The eukaryotic myocilin waspurified from cultured medium of a transformed TM cell line (TM5)transduced with feline immunodeficiency virus that contains an internalcassette expressing full length myocilin. The morphology and adhesion ofhuman TM cells plated either on fibronectin alone or onfibronectin/purified myocilin mixtures were assessed by phase contrastmicroscopy. Actin cytoskeleton was examined using Oregon Greenphalloidin. Immunofluorescence staining for paxillin was alsoperformed.Results: The expression of full length and truncated myocilinproteins in Rosetta2(DE3)pLysS was markedly increased especially whenthe bacteria were grown in media supplemented with 1.0% glucose. Celladhesion was impaired and microspikes were formed when TM cells wereplated onto fibronectin/bacterial full length myocilin mixtures. Loss ofactin stress fibers and focal adhesions was also observed. This myocilinphenotype was also seen with myocilin 1-270, but not with myocilin271-504. The eukaryotic full length myocilin produced nearly identicalde-adhesive effects as those of the bacterially expressed myocilin.Conclusions: The condition for a high level expression of fulllength and truncated myocilins in E. coli was optimized. Thebacterial and eukaryotic recombinant full length myocilin producedsimilar biological consequence on TM cells. The myocilin phenotypeappears to be largely due to the NH2-terminal half of theprotein.

[学科分类] 生物化学/生物物理