Cloning, expression, and partial purification of Rep78: an adeno-associated virus replication protein.
Using the vaccinia virus/T7-RNA polymerase transient protein expression system, the AAV Rep78 protein was expressed in mammalian cells. Rep78 protein was found localized primarily to the nucleus of cells. Maximal steady-state protein levels were reached as early as 12 hr postinfection, with no discernable increase at later time points. The Rep78 protein has been partially purified from nuclear extracts of the expression system. We have successfully used the cloned, purified Rep78 protein to complement an uninfected HeLa cell extract in an in vitro AAV DNA replication assay. Rep78-containing fractions are sufficient to make an uninfected HeLa cell extract competent for AAV DNA replication.
Leonard
C J
CJ
Hearst Microbiology Research Center, Department of Microbiology, Cornell University Medical College, New York, New York 10021.
Berns
K I
KI
D002467:Cell Nucleus / Q000737:chemistry; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D000229:Dependovirus / Q000187:drug effects / Q000254:growth & development*; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D014779:Virus Replication / Q000187:drug effects
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2