Optimized Bacterial Expression of Myocilin Proteins and Functional Comparison of Bacterial and Eukaryotic Myocilins

Abstract

Abstract: : Purpose: To maximize the expression level of myocilin and its truncated proteins in 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 a.a) and two truncated proteins, myocilin 1–270 and myocilin 271–504, were expressed and purified from an E. coli strain, Rosetta2(DE3)pLysS. A transformed TM cell line (TM5) was transduced with feline immunodeficiency virus that contains an internal cassette expressing both wild type myocilin fused to C–terminal His and V5 tags and puromycin N–acetyl–transferase. The eukaryotic myocilin in the TM5 conditioned medium was purified by Ni⁺⁺–affinity chromatography. 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 E. coli was markedly increased when the bacteria were grown in media containing 1.2% glucose followed by IPTG induction. The bacterially expressed full length myocilin produced nearly identical effects on human TM cells as the eukaryotic myocilin. Cell adhesion was impaired and microspikes were formed. Loss of actin stress fibers and focal adhesions was also observed. This myocilin phenotype was more severely seen with myocilin 1–270, but not with myocilin 271–504. Conclusions: The condition for a large–scale expression of full length and truncated myocilins in E. coli was optimized. The bacterial recombinant myocilin produced a similar biological consequence as the protein expressed in an eukaryotic system. The myocilin phenotype may largely be due to the N–terminal half of the protein.

Keywords: protein purification and characterization • proteins encoded by disease genes • trabecular meshwork