Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads.
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.
Oktem
Huseyin Avni
HA
Department of Biological Science, Nanotechnology-Nanobiotechnology Research Center, Middle East Technical University, 06531 Ankara, Turkey.
Bayramoglu
Gulay
G
Ozalp
V Cengiz
VC
Arica
M Yakup
MY
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000502:physiology; D018189:Immunomagnetic Separation / Q000379:methods*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013824:Thermus / Q000201:enzymology* / Q000235:genetics*
D052157:Aptamers, Nucleotide; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase