| dc.contributor.author |
Melissis, S |
en |
| dc.contributor.author |
Labrou, NE |
en |
| dc.contributor.author |
Clonis, YD |
en |
| dc.date.accessioned |
2014-06-06T06:47:10Z |
|
| dc.date.available |
2014-06-06T06:47:10Z |
|
| dc.date.issued |
2006 |
en |
| dc.identifier.issn |
00219673 |
en |
| dc.identifier.uri |
http://dx.doi.org/10.1016/j.chroma.2006.04.044 |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/3432 |
|
| dc.subject |
Affinity chromatography |
en |
| dc.subject |
Biomimetic ligand |
en |
| dc.subject |
Combinatorial chemistry |
en |
| dc.subject |
DNA enzymes |
en |
| dc.subject |
Enzyme purification |
en |
| dc.subject |
Ligand design |
en |
| dc.subject |
Pfu DNA polymerase |
en |
| dc.subject.other |
Adsorption |
en |
| dc.subject.other |
Affinity chromatography |
en |
| dc.subject.other |
Biotechnology |
en |
| dc.subject.other |
DNA |
en |
| dc.subject.other |
Molecular structure |
en |
| dc.subject.other |
Biomimetic ligand |
en |
| dc.subject.other |
Combinatorial chemistry |
en |
| dc.subject.other |
DNA enzymes |
en |
| dc.subject.other |
Enzyme purification |
en |
| dc.subject.other |
Ligand design |
en |
| dc.subject.other |
Pfu DNA polymerase |
en |
| dc.subject.other |
Enzymes |
en |
| dc.subject.other |
DNA |
en |
| dc.subject.other |
DNA directed DNA polymerase alpha |
en |
| dc.subject.other |
DNA directed DNA polymerase beta |
en |
| dc.subject.other |
DNA polymerase |
en |
| dc.subject.other |
nucleotide |
en |
| dc.subject.other |
purine derivative |
en |
| dc.subject.other |
sulfanilic acid |
en |
| dc.subject.other |
sulfonic acid derivative |
en |
| dc.subject.other |
triazine |
en |
| dc.subject.other |
affinity chromatography |
en |
| dc.subject.other |
affinity labeling |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
combinatorial chemistry |
en |
| dc.subject.other |
DNA purification |
en |
| dc.subject.other |
DNA structure |
en |
| dc.subject.other |
DNA synthesis |
en |
| dc.subject.other |
enzyme activity |
en |
| dc.subject.other |
enzyme binding |
en |
| dc.subject.other |
enzyme immobilization |
en |
| dc.subject.other |
enzyme purification |
en |
| dc.subject.other |
priority journal |
en |
| dc.subject.other |
protein motif |
en |
| dc.subject.other |
Pyrococcus furiosus |
en |
| dc.subject.other |
Amino Acid Motifs |
en |
| dc.subject.other |
Amino Acid Sequence |
en |
| dc.subject.other |
Biomimetics |
en |
| dc.subject.other |
Chromatography, Affinity |
en |
| dc.subject.other |
Combinatorial Chemistry Techniques |
en |
| dc.subject.other |
DNA Polymerase I |
en |
| dc.subject.other |
DNA Polymerase II |
en |
| dc.subject.other |
DNA-Directed DNA Polymerase |
en |
| dc.subject.other |
Electrophoresis, Polyacrylamide Gel |
en |
| dc.subject.other |
Escherichia coli |
en |
| dc.subject.other |
Hydrogen-Ion Concentration |
en |
| dc.subject.other |
Ligands |
en |
| dc.subject.other |
Models, Chemical |
en |
| dc.subject.other |
Molecular Sequence Data |
en |
| dc.subject.other |
Molecular Structure |
en |
| dc.subject.other |
Nucleotides |
en |
| dc.subject.other |
Protein Structure, Secondary |
en |
| dc.subject.other |
Pyrococcus furiosus |
en |
| dc.subject.other |
Sepharose |
en |
| dc.subject.other |
Sequence Homology, Amino Acid |
en |
| dc.subject.other |
Escherichia coli |
en |
| dc.subject.other |
Pyrococcus furiosus |
en |
| dc.title |
Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes. One-step purification of Pfu DNA polymerase |
en |
| heal.type |
journalArticle |
en |
| heal.identifier.primary |
10.1016/j.chroma.2006.04.044 |
en |
| heal.publicationDate |
2006 |
en |
| heal.abstract |
The commercial availability of DNA polymerases has revolutionized molecular biotechnology and certain sectors of the bio-industry. Therefore, the development of affinity adsorbents for purification of DNA polymerases is of academic interest and practical importance. In the present study we describe the design, synthesis and evaluation of a combinatorial library of novel affinity ligands for the purification of DNA polymerases (Pols). Pyrococcus furiosus DNA polymerase (Pfu Pol) was employed as a proof-of-principle example. Affinity ligand design was based on mimicking the natural interactions between deoxynucleoside-triphosphates (dNTPs) and the B-motif, a conserved structural moiety found in Pol-I and Pol-II family of enzymes. Solid-phase 'structure-guided' combinatorial chemistry was used to construct a library of 26 variants of the B-motif-binding 'lead' ligand X-Trz-Y (X is a purine derivative and Y is an aliphatic/aromatic sulphonate or phosphonate derivative) using 1,3,5-triazine (Trz) as the scaffold for assembly. The 'lead' ligand showed complementarity against a Lys and a Tyr residue of the polymerase B-motif. The ligand library was screened for its ability to bind and purify Pfu Pol from Escherichia coli extract. One immobilized ligand (oABSAd), bearing 9-aminoethyladenine (AEAd) and sulfanilic acid (oABS) linked on the triazine scaffold, displayed the highest purifying ability and binding capacity (0,55 mg Pfu Pol/g wet gel). Adsorption equilibrium studies with this affinity ligand and Pfu Pol determined a dissociation constant (KD) of 83 nM for the respective complex. The oABSAd affinity adsorbent was exploited in the development of a facile Pfu Pol purification protocol, affording homogeneous enzyme (>99% purity) in a single chromatography step. Quality control tests showed that Pfu Pol purified on the B-motif-complementing ligand is free of nucleic acids and contaminating nuclease activities, therefore, suitable for experimental use. © 2006 Elsevier B.V. All rights reserved. |
en |
| heal.journalName |
Journal of Chromatography A |
en |
| dc.identifier.issue |
1-2 |
en |
| dc.identifier.volume |
1122 |
en |
| dc.identifier.doi |
10.1016/j.chroma.2006.04.044 |
en |
| dc.identifier.spage |
63 |
en |
| dc.identifier.epage |
75 |
en |