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

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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

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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.