UBC Theses and Dissertations
Isolation and characterization of recombinant U24, a membrane protein from human herpesvirus type 6 Tait, Andrew Robert
This thesis describes the isolation and characterization of the U24 membrane protein encoded by Human Herpesvirus type-6 (HHV-6), obtained from an E. coli recombinant expression system. HHV-6 infection has been previously associated with the disease multiple sclerosis (MS), and the U24 protein is of interest because it has a seven amino-acid sequence (PRTPPPS) identical to myelin basic protein (MyBP), a candidate auto-antigen in MS. In the first part of this thesis, I describe the methods that were developed to enable milligram quantities of U24 to be isolated and purified from litre cultures of E. coli. Levels of U24 expressed with a maltose binding protein-hexahistidine fusion tag were particularly enhanced by combinations of low temperature, oxidizing conditions, and/or use of minimal media culture. The significance of these results may be considered useful in application to other difficult-to-obtain membrane proteins. Subsequent chapters of this thesis describe testing the recombinant U24 for potential mimicry of MyBP structure and function. Since the polyproline region in MyBP is now being recognized for its potential in cell-signalling roles that relate to myelin sheath development and structural integrity, I hypothesized that U24 may retain some of the same attributes as MyBP on the basis of identical sequence. Results here suggest that U24 can adopt a polyproline type II helix much like MyBP, which is a structural feature important for engaging in protein-protein interactions. Furthermore, the region is also found to represent a PX(T/S)P MAPK phosphorylation motif and PXXP-based Fyn tyrosine kinase SH3 binding domain. These observations are of particular relevance since phosphorylated MyBP is particularly decreased in MS patients, and Fyn is critical to myelin development. Like MyBP, results suggest that U24 can be phosphorylated at the equivalent threonine and is also able to bind to the Fyn-SH3 domain. These results support the possibility that U24 interferes with essential myelin regulation pathways on the basis of its sequence shared with MyBP, thereby contributing to a pathological process. I conclude with a presentation of preliminary NMR structural data for U24, as well as review results from a study of U24 in an animal model system. Future directions are also discussed.
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