Protein expression, purification, and crystallization of two viral proteins: 1) VP1up from human parvovirus B19 and 2) DNA pilot protein (H) from bacteriophage PhiX174
Abstract
Human parvovirus B19 is the causative pathogen of multiple diseases. Of the two structural proteins encoded by the virus, VP1 and VP2, the unique N-terminus of VP1 (VP1up) is suggested to be a substantial target of the human immune system. Though x-ray and cryo-electron microscopy structures of B19 have been solved, VP1up could not be resolved in these structures. This study was initiated to obtain structural data of VP1up by crystallography, which may be useful for future pharmaceutical research that could block a B19 infection. VP1up protein was expressed in E. coli and purified by chromatography. Matrix crystallization screens were used to screen for VP1up protein crystals; however, crystals have yet to be obtained. Current progress is being made to create VP1up constructs of various lengths. Bacteriophage PhiX174 is a small, icosahedral, E. coli-infecting phage that has been extensively studied by means of genetics, biochemistry, molecular biology, crystallography, and cryo-electron microscopy. Nonetheless, aspects of how this phage proceeds to infect E. coli and replicate its genome remain elusive--with particular attention to the roles of protein H, a structural protein that was not resolved in the crystal structure. This study was initiated to get crystallographic data for protein H, for which initial data had been obtained. Constructs of protein H containing the predicted coiled coil region were expressed, purified, and extensively screened for crystals, which have yet to be discovered. A full length histidine tagged protein H containing a mini-fibritin linker has been expressed and a purification protocol is ongoing.
Degree
M.S.
Advisors
Rossmann, Purdue University.
Subject Area
Virology
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