Protein expression, purification, and crystallization of two viral proteins: 1) VP1up from human parvovirus B19 and 2) DNA pilot protein (H) from bacteriophage PhiX174

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

Proteomic analysis of the membrane skeleton of Tetrahymena thermophila

The membrane skeleton of the Tetrahymena is likely to determine many aspects of the cell surface structure of this ciliated protozoan. The availability of the genomic sequence of Tetrahymena thermophila has facilitated a proteomic analysis of the non-microtubular portion of the cortical cytoskeleton of Tetrahymena thermophila. Potassium iodide-Triton X-100 insoluble residues of these cells retain the cell surface organization of this ciliate, while being depleted in microtubular proteins. Proteins in these membrane skeletal residues have been subsequently fractionated into distinct sets by means of selective extraction and precipitation steps. We have initiated a proteomic analysis of one group of membrane skeletal proteins: the set containing epiplasmin C and associated proteins. Bands containing each of the specific proteins were excised from preparative SDS-polyacrylamide gels. These proteins were reduced, alkylated and digested in-gel with trypsin with the resulting enzymatic peptide fragments analyzed by LC-MS using electrospray ionization. The observed peptide molecular weights were used to search a database of predicted Tetrahymena proteins in order to identify the protein in the original gel band. Further confirmation was obtained by sequencing several enzymatically-derived peptides in the mass spectrometer. Two major components of these structures were identified – epiplasmin C and epiplasmin A – both of which are predicted to be coiled coil proteins. The other major protein to be identified was TCBP-25, an EF-hand containing calcium-binding protein. Several other minor proteins were identified, including epiplasmin B protein, another coiled coil protein. Intriguingly, among the minor proteins present in these preparations were proteins predicted to be related to lipid or protein kinases. Another minor protein was found to be related to a centromeric calcium-binding protein. These minor proteins suggest the possibility of the linkage of these cytoskeletal proteins to cellular signaling processes. We are currently working to analyze protein-protein interactions within the membrane skeleton by means of non-denaturing gel electrophoresis of protein complexes, as well as far-Western blotting experiments