Expression and purification of the HIV-1 reverse transcriptase using the baculovirus expression vector system.
We have successfully expressed and purified the human immunodeficiency virus type-1 reverse transcriptase (RT) using the baculovirus expression vector system. This expression system provides a eukaryotic environment in which post-translational modifications of foreign gene products can occur. After infection with recombinant virus, Western blot analysis confirmed the presence of an immunoreactive polypeptide of approximately 66 kDa from insect Sf9 cell lysates. RT was then purified from crude extracts of baculovirus-infected Sf9 cells; SDS-PAGE analysis of fractions obtain from partial purification showed that in contrast to the Escherichia coli-expressed RT, the baculovirus-expressed RT corresponded to a doublet of peptides at approximately 66 kDa. Further purification of the protein resulted in a p66 protein, judged to be more than 90% pure by SDS-PAGE and Coomassie blue stain. Following purification, the baculovirus derived RT had specific activity for DNA polymerase similar to that of the E. coli-derived RT. Therefore, RT purified from Sf9 cells appears to be suitable for structure-function studies of this enzyme.
Kawa
S
S
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0645.
Kumar
A
A
Smith
J S
JS
Becerra
S P
SP
Beard
W A
WA
Wilson
S H
SH
Thompson
E B
EB
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics*; D008970:Molecular Weight; D009036:Moths; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection
D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase