Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli.
Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli resulted in the synthesis of reverse transcriptase activity which could be detected in crude extracts. Construction of deletions at the 3' terminus of this gene resulted in a 4-fold increase in the level of the reverse transcriptase activity in the soluble fraction of crude lysates and yielded the high level production of a stable protein species of Mr = 71,000. Purification of this protein by column chromatography on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite indicated that it was a multifunctional enzyme containing RNase H and reverse transcriptase activity. The Mr = 71,000 species had a sedimentation coefficient of 4.65 S by glycerol gradient centrifugation, indicating that the enzyme was a monomer. Using poly(A)+ mRNAs primed with oligo(dT), the enzyme synthesized double-stranded DNA copies between 1.3 and 9.9 kilobases in length. Synthesis of long cDNA required 8 mM Mg2+, 4 mM Mn2+, 2 mM dNTPs, and saturating levels of enzyme. Actinomycin D efficiently limited the enzyme to the first strand synthesis. Additional characteristics of the fusion protein are described.
Roth
M J
MJ
Tanese
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N
Goff
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D002872:Chromosome Deletion; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D005333:Fetus; D005796:Genes; D005814:Genes, Viral; D006801:Humans; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D009132:Muscles / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011247:Pregnancy; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification*; D013698:Templates, Genetic
D012194:RNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes