Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes One-step purification of Pfu DNA polymerase.
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 (K(D)) 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.
Melissis
S
S
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece.
Labrou
N E
NE
Clonis
Y D
YD
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D032701:Biomimetics / Q000379:methods; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004257:DNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D008024:Ligands; D009711:Nucleotides; D012685:Sepharose; C072146:Pfu DNA polymerase; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase