Quick and easy method for extraction and purification of Pfu-Sso7d, a high processivity DNA polymerase.

Autor: Farooqui AK; Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India., Ahmad H; Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India., Rehmani MU; Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India., Husain A; Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India. Electronic address: afzal.bc@amu.ac.in.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2023 Aug; Vol. 208-209, pp. 106276. Date of Electronic Publication: 2023 May 06.
DOI: 10.1016/j.pep.2023.106276
Abstrakt: The polymerase chain reaction is an extensively used technique with numerous applications in the field of biological sciences. In addition to naturally occurring DNA polymerases with varying processivity and fidelity, genetically engineered recombinant DNA polymerases are also used in PCR. The Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA binding protein, to the polymerase domain of the Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity. Expensive commercial variants of Pfu-Sso7d are sold under various trade names. Here, we report a quick, cost and time-efficient purification protocol and an optimized buffer system for Pfu-Sso7d. We evaluated precipitation efficiencies of varying concentrations of ethanol and acetone and compared the activities of the precipitated enzyme. Although both the solvents efficiently precipitated Pfu-Sso7d, acetone showed better precipitation efficiency. Purified Pfu-Sso7d showed excellent activity in the PCR of templates with varying lengths and GC contents. We also report a buffer system that works with Pfu-Sso7d as efficiently as commercially available buffers. This quick and efficient purification scheme and buffer system will provide researchers cost-efficient access to fusion polymerase.
Competing Interests: Declaration of competing interest The authors declare no competing financial or non-financial interests.
(Copyright © 2023 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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