One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography

dc.contributor.author	Melissis, S	en
dc.contributor.author	Labrou, NE	en
dc.contributor.author	Clonis, YD	en
dc.date.accessioned	2014-06-06T06:48:01Z
dc.date.available	2014-06-06T06:48:01Z
dc.date.issued	2007	en
dc.identifier.issn	18606768	en
dc.identifier.uri	http://dx.doi.org/10.1002/biot.200600191	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/3919
dc.subject	Affinity chromatography	en
dc.subject	Biomimetic ligand	en
dc.subject	Combinational chemistry	en
dc.subject	Ligand design	en
dc.subject	Taq DNA polymerase	en
dc.subject.other	adenosine triphosphate	en
dc.subject.other	aniline	en
dc.subject.other	ligand	en
dc.subject.other	nuclease	en
dc.subject.other	nucleotide	en
dc.subject.other	parathion	en
dc.subject.other	Taq polymerase	en
dc.subject.other	triazine	en
dc.subject.other	biomimetic material	en
dc.subject.other	adsorption	en
dc.subject.other	affinity chromatography	en
dc.subject.other	article	en
dc.subject.other	cell lysate	en
dc.subject.other	combinatorial library	en
dc.subject.other	dissociation constant	en
dc.subject.other	enzyme activity	en
dc.subject.other	enzyme purification	en
dc.subject.other	Escherichia coli	en
dc.subject.other	nonhuman	en
dc.subject.other	nucleotide sequence	en
dc.subject.other	priority journal	en
dc.subject.other	screening	en
dc.subject.other	chemistry	en
dc.subject.other	enzymology	en
dc.subject.other	isolation and purification	en
dc.subject.other	methodology	en
dc.subject.other	protein binding	en
dc.subject.other	Thermus aquaticus	en
dc.subject.other	Biomimetic Materials	en
dc.subject.other	Chromatography, Affinity	en
dc.subject.other	Escherichia coli	en
dc.subject.other	Nucleotides	en
dc.subject.other	Protein Binding	en
dc.subject.other	Taq Polymerase	en
dc.title	One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography	en
heal.type	journalArticle	en
heal.identifier.primary	10.1002/biot.200600191	en
heal.publicationDate	2007	en
heal.abstract	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 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 (rnABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) 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 (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu 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 mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.	en
heal.journalName	Biotechnology Journal	en
dc.identifier.issue	1	en
dc.identifier.volume	2	en
dc.identifier.doi	10.1002/biot.200600191	en
dc.identifier.spage	121	en
dc.identifier.epage	132	en