Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads

Date

2007-01-01

Author

Öktem, Hüseyin Avni
Ozalp, V. Cengiz
Arica, M. Yakup

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

162
views

0
downloads

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract.

Subject Keywords

Biotechnology

URI

https://hdl.handle.net/11511/36166

Journal

BIOTECHNOLOGY PROGRESS

DOI

https://doi.org/10.1021/bp0602505

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment Hakki, EE; Akkaya, Mahinur (Elsevier BV, 2001-02-01) No amino acid or DNA sequence information in sequence databases was found for a fungal lactate dehydrogenase (LDH) isozyme. Highly conserved regions in the lactate dehydrogenase enzymes of all taxonomies are found to be beta alpha beta nucleotide binding and substrate binding sites, also catalysis/active site. The conserved regions were selected as PCR primer target regions. The degenerate primers were designed according to the codon usage, determined by analyzing a number of different genes of Rhizopus spe...
Development of PCR methods for detection and quantification of genetically modified maize Jabbari Farhoud, Houman; Gültekin, Güzin Candan; Department of Biotechnology (2010) This study describes development of methods for screening, identification and quantification of genetic modifications in maize samples. Totally 88 maize samples were collected randomly throughout Turkey in three years from 2006 to 2008 and were analyzed. Two maize samples that were detected as GM positive in previous studies were selected as positive controls. Following the DNA extraction by manual CTAB method, conventional PCR methods were employed for screening of genetic modifications in samples by detec...
A rapid and simple method for staining of the crystal protein of bacillus thuringiensis Sharif, Fade A.; Alaeddinoglu, Naif Gürdal (Springer Science and Business Media LLC, 1988-6) A rapid and simple method of staining for the crystal protein (δ-endotoxin or parasporal body) ofBacillus thuringiensis has been developed. Changes in colonial morphology were observed when cells lost their ability to form crystal protein or both crystal protein and spore.
Isolation and characterization of Taq DNA polymerase and optimization and validation of newly designed thermal cyclers Yıldız, Lütfiye; Öktem, Hüseyin Avni; Bilecen, Kıvanç; Department of Biotechnology (2011) Amplification of target DNA in vitro via polymerase chain reaction (PCR) is a widely used scientific technique in molecular biology. This method relies on repeated heating and cooling cycles of the DNA and enzyme mixture, resulting with the enzymatic replication of the DNA. A heat stable Taq DNA polymerase and a thermal cycler that enables repeated heating/cooling cycles are the two key components of the PCR. In this study we have produced a high activity Taq DNA polymerase and used this enzyme to validate ...
Purification, characterization, crystallization and preliminary x-ray structure determination of scytalidium thermophilum bifunctional catalase and identification of its catechol oxidase activity Sutay, Didem; Bakır, Ufuk; Department of Chemical Engineering (2007) In this study, the aim was identification and classification of the enzyme having phenol oxidase activity produced by a thermophilic fungus, Scytalidium thermophilum. For this purpose, enzyme production, purification, biochemical characterization and structural analysis by X-ray crystallography studies have been performed. At the beginning of the research, this enzyme was considered as a phenol oxidase and analyzed accordingly. However, during purification, amino acid sequencing and structural studies, the ...

Citation Formats

H. A. Öktem, V. C. Ozalp, and M. Y. Arica, “Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads,” BIOTECHNOLOGY PROGRESS, pp. 146–154, 2007, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/36166.