This chapter describes the purification of RNA-dependent DNA polymerase from avian myeloblastosis virus (AMV). The reverse transcriptase has been purified from AMV approximately 80- to 100-fold over the activity detected in crude ammonium sulfate fractions, yielding preparations capable of incorporating into DNA more than 3.1 μmoles of deoxynucleotide per milligram of protein in 30 minutes. During the course of this purification, the yield of DNA synthesized in reactions primed with AMV RNA decreased with each purification step. A small molecular weight protein, referred to as stimulatory protein, can be isolated during the phosphocellulose chromatography step. When added to purified polymerase preparations, this protein increases the yield of RNA-primed DNA synthesis. The stimulatory protein increases the rate and yield of DNA synthesized in reactions containing viral RNA and purified viral polymerase. The amount of stimulatory protein giving optimal stimulation must be determined with each system because high concentrations are inhibitory.
ASJC Scopus subject areas
- Molecular Biology