Purification of M-MLVH- RT on a 9-aminoethyladenine-(1,6-diamine-hexane)-triazine selected from a combinatorial library of dNTP-mimetic ligands.
Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH- RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH- RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions (6 mM MnCl(2) in 20 mM Tris-HCl buffer, pH 7.5). The binding capacity of immobilized AEAd with M-MLVH- RT was determined to be equal to approximately 1 mg enzyme/g moist weight gel. Adsorption studies with immobilized AEAd and soluble M-MLVH- RT demonstrated that the formation of the respective complex was perturbed by ATP. Quality control tests of the purified M-MLVH- RT essentially showed a single band (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and absence of nucleic acids and contaminating nuclease activities.
Melissis
Sotirios C
SC
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece.
Papageorgiou
Anastassios C
AC
Labrou
Nikolaos E
NE
Clonis
Yannis D
YD
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000327:Adsorption; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D056004:Molecular Dynamics Simulation; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014227:Triazines / Q000737:chemistry / Q000378:metabolism*
D003854:Deoxyribonucleotides; D011994:Recombinant Proteins; D014227:Triazines; D000255:Adenosine Triphosphate; D012194:RNA-Directed DNA Polymerase