Application of protein expression and purification to study virus fusion and mammalian transcription factors

Fundamental questions in the study of human cytomegalovirus (HCMV) fusion and mammalian transcription factors, Nrf2 and CARM1, can be effectively addressed through application of protein purification methodologies. HCMV is an important pathogen affecting immunocompromised patients for which there are few safe therapies. The virus utilizes a complex fusion mechanism for entry into cells. This work describes the purification of a viral glycoprotein B fragment and the characterization of its fusion-inhibiting activity. The characterization was used to develop potent beta-peptide based mimetics that specifically inhibited virus fusion. The potency of the short beta-peptides (IC 50=28 microM) was a 10-fold improvement over previous peptide-based inhibitors of HCMV Type III fusion mechanism.;The Nrf2 transcription factor has been shown to affect various neurodegenerative human diseases through activation of the antioxidant response element (ARE). Nrf2 when activated, effectively mobilizes endogenous protective machinery to combat oxidative stress caused by reactive oxygen species (ROS). Protection assays in primary neuronal cultures demonstrated that activation of Nrf2 protects neurons against in vitro toxicity of amyloid beta (Abeta) peptides. The neuronal protection activity depended on the abundance of co-cultured astrocytes. A panel of monoclonal antibodies against recombinant mouse Nrf2 was generated. The antibodies were found to be efficacious in ELISA, western blotting, and immunofluorescence assays. The protein transduction domain TAT has been shown to stimulate uptake of fused cargo proteins. A TAT-Nrf2 fusion protein was purified to homogeneity and refolded from denatured inclusion bodies. The TAT-Nrf2 chimera was shown to protect cultured neuronal cell against ROS-mediated cytotoxicity by activation of ARE-dependent genes. The CARM1 transcription factor is a member of protein arginine methyltransferase family and has been implicated in breast and prostate cancer. CARM1 not only methylates diverse substrates important for DNA and RNA processing but also methylates itself. Previous studies of CARM1 automethylation activity were hampered by lack of good protein purification methodologies. This work describes the utilization of HaloTag(TM) technology for purification of soluble, full-length, enzymatically active CARM1 protein from inclusion bodies in E. coli and from mammalian HEK293T cells. The purified protein was used to identify the automethylation site of CARM1 and characterize its effect on substrate methylation.