Separation of cellular and viral DNA polymerases by affinity chromatography on polynucleotide-Sepharose.
Polyguanylate- and poly(2'-O-methyl)uridylate-Sepharose have been prepared for affinity chromatography of DNA polymerases of viral origin (reverse transcriptase). Both cellular DNA polymerases and reverse transcriptase bind to polyguanylate-Sepharose. The cellular polymerases can be eluted from the column between 0.32 and 0.42 M NaCl while reverse transcriptase eluted between 0.56 and 0.78 M NaCl. However, only reverse transcriptase adheres to poly(2'-O-methyl)uridylate-Sepharose and can be eluted at approximately 0.35 M NaCl. The columns were used to partially purify RNA-dependent DNA polymerase from spleens of mice infected with Rauscher leukemia virus. The enzyme preparation is about 1300-fold purified and is inhibited by antiserum prepared against purified reverse transcriptase from Rauscher leukemia virus to the same extent as the virion enzyme.更多