Functional reverse transcriptase encoded by the human LINE-1 from baculovirus-infected insect cells.
The human LINE-1 ORF2, which encodes reverse transcriptase, was inserted into a baculovirus shuttle vector and expressed in Sf 21 cells. An immunoreactive polypeptide (149kDa) synthesized by infected cells had reverse transcriptase activity. A procedure for purification of functional ORF2 protein from insect cells was developed. The enzyme was purified with good recovery to near homogeneity and retained stable DNA polymerase activity. The optimum reaction conditions of the enzyme were determined with respect to salts, pH, and temperature. Substrate specificities and divalent cation requirements were investigated. The recombinant enzyme had a 3-fold preference for Mg2+ over Mn2+ for reverse transcriptase activity on poly(rA).oligo(dT)(12). As for DNA synthesis, the recombinant ORF2 protein was found to possess both RNA-dependent and DNA-dependent DNA polymerase activities.
Piskareva
Olga
O
Department of Molecular Biology, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Moscow Region 142290, Russia.
Denmukhametova
Svetlana
S
Schmatchenko
Vadim
V
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase