PURIFICATION OF RECOMBINANT PFU DNA POLYMERASE BY DOUBLE STEP AFFINITY CHROMATOGRAPHY
Main Article Content
Authors
A. Mussakhmetov
National Center for Biotechnology, Valikhanov str. 13/1, Astana, 010000, Kazakhstan
A. Nurmagambetova
National Center for Biotechnology, Valikhanov str. 13/1, Astana, 010000, Kazakhstan
S. Abeldenov
National Center for Biotechnology, Valikhanov str. 13/1, Astana, 010000, Kazakhstan
B. Khassenov
National Center for Biotechnology, Valikhanov str. 13/1, Astana, 010000, Kazakhstan
Abstract
In Kazakhstan, the production of enzymes for molecular biology is not established. This explains the absence of any high-tech production and low demand for this specific product. However, PCR diagnostics and genomic technologies in Kazakhstan is already a reality, and the need for thermostable polymerases in the country is growing up.
Recombinant Pfu DNA polymerase was purified from Escherichia coli. Expression vector with cloned gene coding Pfu DNA polymerase protein was made by standard genetic engineering methods. Recombinant protein has 20 additional amino acids at N-end and has a molecular mass of 92,3 kDa. Results of this work showed that PCR made with recombinant analog of Pfu DNA polymerase yields amplification products of different DNA sequence length. In addition, the experiment for thermostability was done and enzyme showed polymerase activity after several hours of heating.
Keywords
Pfu DNA polymerase, polymerase chain reaction, Pyrococcus furiosus, recombinant protein
Article Details
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