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One‐step purification of Taq DNA polymerase using nucleotide‐mimetic affinity chromatography
Author(s) -
Melissis Sotirios,
Labrou Nikolaos E.,
Clonis Yannis D.
Publication year - 2007
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200600191
Subject(s) - thermus aquaticus , affinity chromatography , chromatography , taq polymerase , chemistry , nuclease , dna polymerase , polymerase , dna , enzyme , biochemistry
The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure‐biased combinatorial library of dNTP‐mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand ( m ABSGu) of the general formula X‐Trz‐Y, bearing 9‐aminoethylguanine (AEGu) and aniline‐2‐sulfonic acid ( m ABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant ( K D) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the m ABSGu‐Taq Pol complex. The m ABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, ∼61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the m ABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities.