| dc.contributor.author |
Melissis, SC |
en |
| dc.contributor.author |
Papageorgiou, AC |
en |
| dc.contributor.author |
Labrou, NE |
en |
| dc.contributor.author |
Clonis, YD |
en |
| dc.date.accessioned |
2014-06-06T06:50:39Z |
|
| dc.date.available |
2014-06-06T06:50:39Z |
|
| dc.date.issued |
2010 |
en |
| dc.identifier.issn |
00219665 |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/5107 |
|
| dc.relation.uri |
http://www.scopus.com/inward/record.url?eid=2-s2.0-77955504085&partnerID=40&md5=2d0d401f9237c7c567085eaa74113f1f |
en |
| dc.subject.other |
adenosine triphosphate |
en |
| dc.subject.other |
deoxyribonucleotide |
en |
| dc.subject.other |
recombinant protein |
en |
| dc.subject.other |
RNA directed DNA polymerase |
en |
| dc.subject.other |
triazine derivative |
en |
| dc.subject.other |
adsorption |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
chemistry |
en |
| dc.subject.other |
combinatorial chemistry |
en |
| dc.subject.other |
enzymology |
en |
| dc.subject.other |
Escherichia coli |
en |
| dc.subject.other |
genetics |
en |
| dc.subject.other |
isolation and purification |
en |
| dc.subject.other |
metabolism |
en |
| dc.subject.other |
methodology |
en |
| dc.subject.other |
molecular dynamics |
en |
| dc.subject.other |
Moloney leukemia oncovirus |
en |
| dc.subject.other |
pH |
en |
| dc.subject.other |
polyacrylamide gel electrophoresis |
en |
| dc.subject.other |
protein binding |
en |
| dc.subject.other |
Adenosine Triphosphate |
en |
| dc.subject.other |
Adsorption |
en |
| dc.subject.other |
Combinatorial Chemistry Techniques |
en |
| dc.subject.other |
Deoxyribonucleotides |
en |
| dc.subject.other |
Electrophoresis, Polyacrylamide Gel |
en |
| dc.subject.other |
Escherichia coli |
en |
| dc.subject.other |
Hydrogen-Ion Concentration |
en |
| dc.subject.other |
Molecular Dynamics Simulation |
en |
| dc.subject.other |
Moloney murine leukemia virus |
en |
| dc.subject.other |
Protein Binding |
en |
| dc.subject.other |
Recombinant Proteins |
en |
| dc.subject.other |
RNA-Directed DNA Polymerase |
en |
| dc.subject.other |
Triazines |
en |
| dc.subject.other |
Escherichia coli |
en |
| dc.subject.other |
Moloney murine leukemia virus |
en |
| dc.title |
Purification of M-MLVH-RT on a9-aminoethyladenine-(1,6-diamine-hexane)- triazine selected from a combinatorial library of dNTP-mimetic ligands |
en |
| heal.type |
journalArticle |
en |
| heal.publicationDate |
2010 |
en |
| heal.abstract |
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 MnCl2 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. |
en |
| heal.journalName |
Journal of Chromatographic Science |
en |
| dc.identifier.issue |
6 |
en |
| dc.identifier.volume |
48 |
en |
| dc.identifier.spage |
496 |
en |
| dc.identifier.epage |
502 |
en |