Improved factor Xa cleavage of fusion proteins containing maltose binding protein.
(1995)
Journal - BioTechniques (UNITED STATES )
Abstract :
The addition of five glycine residues at a position adjacent to the factor Xa cleavage site of a MBP-2C fusion protein, comprising maltose binding protein (MBP) and poliovirus 2C, allowed factor Xa to generate both of the component proteins. If, however, MBP-2C was without the above modification, it was cleaved only at a site located internally within poliovirus 2C, and this protein was not, therefore, generated by factor Xa cleavage. A simple procedure is described that uses PCR for the introduction of five glycines adjacent to the factor Xa recognition site.
| ISSN : | 0736-6205 |
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| Mesh Heading : | Amino Acid Sequence Base Sequence Blotting, Western Carrier Proteins Factor Xa Glycine Molecular Sequence Data Peptide Hydrolases Plasmids Viral Fusion Proteins Viral Nonstructural Proteins chemistry chemistry chemistry chemistry |
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| Mesh Heading Relevant : | chemistry chemistry chemistry |
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Nonradioactive northwestern analysis using biotinylated riboprobes.
(1995)
Journal - BioTechniques (UNITED STATES )
Abstract :
A nonradioactive modification of the Northwestern assay is described and applied to the detection of RNA-binding proteins. The nonradioactive assay is based on the use of biotinylated riboprobes, which are stable and easy to handle. Chemiluminescence is generated with streptavidin-conjugated peroxidase and provides even better sensitivity than the radioactive detection method to assay RNA binding to proteins bound to nitrocellulose.
| ISSN : | 0736-6205 |
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| Mesh Heading : | Biotin RNA-Binding Proteins |
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| Mesh Heading Relevant : | RNA Probes analysis |
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Poliovirus protein 2C has ATPase and GTPase activities.
(1993)
Journal - The Journal of biological chemistry (UNITED STATES )
Abstract :
Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids.
| ISSN : | 0021-9258 |
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| Mesh Heading : | Adenosine Triphosphatases Base Sequence Carrier Proteins Cloning, Molecular Escherichia coli GTP Phosphohydrolases Molecular Sequence Data Mutagenesis Oligodeoxyribonucleotides Plasmids Poliovirus RNA Helicases RNA Nucleotidyltransferases RNA, Double-Stranded Recombinant Fusion Proteins Sequence Deletion Viral Nonstructural Proteins genetics isolation & purification genetics isolation & purification genetics genetics isolation & purification genetics metabolism metabolism isolation & purification metabolism genetics isolation & purification |
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| Mesh Heading Relevant : | ATP-Binding Cassette Transporters Escherichia coli Proteins Monosaccharide Transport Proteins metabolism metabolism metabolism metabolism metabolism |
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Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol.
(1992)
Journal - Journal of virology (UNITED STATES )
Abstract :
The mode of action of gliotoxin against poliovirus has been analyzed in detail. This fungal metabolite inhibits the appearance of poliovirus proteins when present from the beginning of infection but has no effect on viral translation when added at late times. In agreement with previous findings, this toxin potently inhibited the incorporation of [3H]uridine into poliovirus RNA soon after its addition to the culture medium. Analysis of the synthesis of poliovirus plus- or minus-stranded RNA in the presence of gliotoxin suggests that this compound effectively hampered both processes. This result contrasts with the mode of action of other inhibitors of poliovirus RNA synthesis, such as guanidine or flavones, that selectively block plus-stranded RNA synthesis and suggests that the target of gliotoxin differs from the target of guanidine, i.e., poliovirus protein 2C. Indeed, gliotoxin was found to be a potent inhibitor of poliovirus RNA synthesis in cell-free systems, using membranous crude replication complexes, a reaction that is not blocked by guanidine or Ro 09-0179. Moreover, in vitro activity of the purified poliovirus polymerase 3Dpol was efficiently inhibited by gliotoxin. These results indicate that this toxin acts on the poliovirus polymerase 3Dpol, providing the first description of an inhibitor of this viral enzyme.
| ISSN : | 0022-538X |
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| Mesh Heading : | Blotting, Northern DNA-Directed RNA Polymerases Gliotoxin Hela Cells Humans Kinetics Poliovirus Protein Biosynthesis RNA, Viral drug effects drug effects |
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| Mesh Heading Relevant : | antagonists & inhibitors pharmacology genetics biosynthesis |
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