In-house Extraction and Purification of Pfu-Sso7d, a High-processivity DNA Polymerase.

ABSTRACT

The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase. Key features • We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d. • Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance. • Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers. • The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCR applications.