PMID: 9445385Mar 7, 1998Paper

Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization

The Biochemical Journal
R TaubeAmnon Hizi

Abstract

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific r...Continue Reading

Citations

Apr 13, 2013·International Journal of Cancer. Journal International Du Cancer·Brian Salmons, Walter H Gunzburg
Nov 14, 2014·Environmental Microbiology Reports·María Gómez-LozanoSøren Molin
May 25, 2002·The Journal of General Virology·Sharon K Fodor, Volker M Vogt
Jan 4, 2017·Virus Research·Luis Menéndez-AriasMar Álvarez
Apr 2, 2010·Cellular and Molecular Life Sciences : CMLS·Alon Herschhorn, Amnon Hizi
Jul 1, 2009·Nature Methods·Sam E V LinsenEdwin Cuppen

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