This EV71 Enterovirus 71 VP0 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of Enterovirus 71 VP0 protein (Cat: 40013-V20B) from the overexpression lysate was verified.
A DNA sequence encoding the amino acids (Met 1-Gln 323) of human enterovirus 71 genome polyprotein (Q66478-1), corresponding to the protein VP0 precusor, a component of immature procapsids, was fused with the N-terminal polyhistidine-tagged GST tag at the N-terminus.
The recombinant human enterovirus 71 protein VP0/GST chimera consists of 560 amino acids and has a calculated molecular mass of 63 kDa. It migrates as an approximately 58 kDa band in SDS-PAGE under reducing conditions.
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Human enterovirus 71 genome polyprotein is a member of the picornaviruses polyprotein family. It contains two peptidase C3 domains, one RdRp catalytic domain, one SF3 helicase domain. Genome polyprotein is cleaved into the following 12 chains: Protein VP (VP4-VP2), Protein VP4 (P1A), Protein VP2 (P1B), Protein VP3 (P1C), Protein VP1 (P1D), Picornain 2A (P2A), Protein 2B (P2B), Protein 2C (P2C), Protein 3A (P3A), Protein 3B (P3B), Picornain 3C (Protease 3C) and RNA-directed RNA polymerase 3D-POL (P3D-POL). VP precursor is a component of immature procapsids. Capsid proteins VP1, VP2, VP3 and VP4 form a closed capsid enclosing the viral positive strand RNA genome. VP4 lies on the inner surface of the protein shell formed by VP1, VP2 and VP3. All the three latter proteins contain a beta-sheet structure called beta-barrel jelly roll. Together they form an icosahedral capsid composed of 6 copies of each VP1, VP2, and VP3, with a diameter of approximately 3 Angstroms. VP1 is situated at the 12 fivefold axes, whereas VP2 and VP3 are located at the quasi-sixfold axes.
Brown B.A., et al., 1995, Virus Res. 39:195-206.
Tang W.-F. et al., 2007, J. Biol. Chem. 282:5888-5898.
Huang,S.C. et al., 2008, Virus Res. 131 (2):250-9.