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


Oktem H. A. , Bayramoglu G., Ozalp V. C. , Arica M. Y.

BIOTECHNOLOGY PROGRESS, vol.23, no.1, pp.146-154, 2007 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 23 Issue: 1
  • Publication Date: 2007
  • Doi Number: 10.1021/bp0602505
  • Title of Journal : BIOTECHNOLOGY PROGRESS
  • Page Numbers: pp.146-154

Abstract

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.