Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Processing of the envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV) was investigated by using cDNA clones covering the structural and part of the nonstructural (NS) protein regions. The cDNA clones expressed in mammalian and insect cells were immunoprecipitated by serum of a hepatitis C patient and by monoclonal and polyclonal antibodies raised against the recombinant proteins expressed in insect cells or Escherichia coli. The E2 protein expressed in both insect and mammalian cells was a glycoprotein of 60 kDa (gp60) and removal of the sugar residues by N-glycanase yielded 38- and 40-kDa proteins. Pulse-chase experiments revealed that efficient expression and processing of the envelope proteins required coexpression with the flanking core and NS2 proteins. Not only E1 and E2 proteins but also NS2 and NS3 proteins were coprecipitated by anti-E1 or anti-E2 monoclonal antibody in the cells infected with the recombinant baculovirus expressing structural and NS proteins (NS2 and NS3), while only the NS3 protein was precipitated by anti-NS3 antibody. The association of E1 and E2 proteins was not influenced by the presence of a reducing agent and was still observed in the cells coinfected with the deletion mutants lacking both internal and C-terminal hydrophobic regions of each protein. Furthermore, the truncated forms of the E1 and E2 proteins were secreted into the culture supernatant and some of them were still associated with each other.
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PMID:Processing of E1 and E2 glycoproteins of hepatitis C virus expressed in mammalian and insect cells. 797 9

Development of heterologous systems to produce useful HCV vaccine candidates is an important part of HCV research. In this study different HCV structural region variants were designed to express the first 120 aa, 176 aa, 339 aa, and 650 aa of HCV polyprotein, and aa 384 to 521, or aa 384-605 or aa 384-746 of HCV E2 protein fused to the leader sequence of sucrose invertase 2 allowing the secretion of recombinant E2 proteins. Low expression levels were observed for HCV core protein (HCcAg) variants expressing the first 120 aa and 176 aa (HCcAg.120 and HCcAg.176, respectively). Higher expression levels were observed when HCcAg was expressed as a polypeptide with either E1 or E1 and E2 proteins. In addition, HCcAg was processed to produce two antigenic bands with 21 and 23kDa (P21 and P23, respectively) when expressed as a polypeptide with HCV E1 and E2 proteins. Results also suggest E1 processing in the context of HCcAg.E1.E2 polyprotein. On the other hand, E2.521, E2.605, and E2.680 were efficiently excreted to the culture medium. However, the entire E2.746 variant predominantly localized in the insoluble fraction of ruptured cells. Results suggest that the hydrophobic C-terminal E2 region from aa 681 to 746 is critical for intracellular retention of recombinant E2.746 protein in Pichia pastoris cells. Endo H or PNGase F treatment suggests that E2.746 was modified with high-mannose type oligosaccharides in P. pastoris. These data justify the usefulness of P. pastoris expression system to express HCV structural viral proteins which may be useful targets for HCV vaccine candidates.
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PMID:Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast. 1648 93

The E2 envelope glycoprotein of virulent Shimen strain and avirulent C-strain of Classical swine fever virus (CSFV) has 5 and 6 potential glycosylation sites, respectively, and the potential glycosylation site 986N is unique to C-strain. To study the differences in glycosylation between the virus pair, the E2 genes (removing signal sequence and transmembrane anchor regions) of the two strains fused with the melittin signal sequence were expressed in the Sf9 insect cells. The recombinant E2 proteins were secreted into the medium of Sf9 cells in dimer form with different molecular weight (MW). Deglycosylation of the recombinant E2 proteins by endo H and PNGase F showed that the deglycosalated Shimen-E2 and HCLV-E2 have the same MW, indicating that the different MW between Shimen-E2 and HCLV-E2 proteins came from different glycosylation. Site-directed mutagenesis in the potential glycosylation site at 986N demonstrated that the mutated Shimen-E2 protein had the same MW as the wild-type HCLV-E2 protein, while the mutated HCLV-E2 had the same MW as the wild-type Shimen-E2 protein. We suggest that the different MW between Shimen-E2 and HCLV-E2 is resulted from the different glycosylation on 986 N glycosylation site.
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PMID:[Differences in glycosylation of the E2 protein between virulent Shimen strain and a virulent C-strain of classical swine fever virus]. 1796 57