Purification and characterization of an active form of the p78Rep protein of adeno-associated virus type 2 expressed in Escherichia coli.
The 78-kDa product (p78Rep) of the rep gene of AAV-2 was expressed with an amino-terminal histidine-tag in Escherichia coli and was purified under denaturing conditions. After renaturation of the p78Rep protein by serial steps of dialysis, the biochemical activities of the p78Rep protein were demonstrated, which include the ATP-dependent endonuclease and helicase activity as well as sequence-specific binding to the AAV-2 terminal repeat. These activities were retained when the protein was purified under denaturing conditions followed by renaturation. When compared with published data for p68Rep, the helicase activity of p78Rep was stronger and the endonuclease activity was weaker. The p78Rep protein was able to inhibit HIV-1 replication after co-microinjection together with infectious proviral HIV-1 DNA into the nuclei of human cells, suggesting that p78Rep is necessary for inhibition of HIV-1 in vivo.
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Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany.
Frey
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M
Zentgraf
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H
Sczakiel
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G
D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004265:DNA Helicases / Q000096:biosynthesis* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D014779:Virus Replication
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D004265:DNA Helicases