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Summer Research Fellowship Programme of India's Science Academies

Extensive study on isolation and purification of phusion polymerase

Nandhana Eswaran

B.Tech, Anna University, Chennai 600 025

Dr. Mahak Sharma

Assistant professor, Department of biological sciences, IISER Mohali, Manauli.

Abstract

Phusion polymerase is a chimeric protein obtained by combining the genes encoding pfu polymerase with a DNA binding domain. The resulting protein will thus have enhanced processivity along with proof-reading ability, making it suitable for all PCR applications requiring long amplicons.

A simple and rapid method for extracting DNA polymerase from hyperthermophilic bacteria, Pyrococcus furiosus, is presented here. The purification procedure involved the use of heat and chromatographic methods like ion-exchange and affinity based techniques. The extracts were loaded onto a sodium dodecyl sulfate-polyacrylamide electrophoresis gel to assay the purity level. The molecular weight of the phusion protein was found to be about 97KDa. The activity was checked by using PCR reactions.

Keywords: phusion polymerase, protein purification.

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