Purification of Recombinant Pol γA and Pol γB

Proteins were prepared as previously described (13 (link)). Briefly, Pol γA WT, Exo-deficient [D198A/E200A (exo⁻)], and mutants [Pol γA R853A, Pol γA R853Q, Pol γA R853W, and Pol γA R853A (exo⁻)] were expressed in insect cells Sf9 and purified using TALON (Clontech) and Superdex 200 (Cytiva) column chromatography. Pol γB WT and a deletion mutant Pol γB-ΔI4 (deletion of amino acids 136 to 182) were expressed in E. coli Rosetta BL21 DE3 and purified using Nickel-NTA (Qiagen) and cation-exchange (Mono S) chromatography. For structural studies, purified Pol γA and Pol γB were mixed at a 1:2 molar ratio on ice for 10 min and purified using Superdex 200 (Cytiva) column chromatography. The protein purity was estimated to be >95% per SDS–polyacrylamide gel electrophoresis analysis.

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Park J., Herrmann G.K., Roy A., Shumate C.K., Cisneros G.A, & Yin Y.W. (2024). An interaction network in the polymerase active site is a prerequisite for Watson-Crick base pairing in Pol γ. Science Advances, 10(21), eadl3214.

Publication 2024

Superdex 200 Nickel-NTA

Corresponding Organization : The University of Texas at Dallas

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Variable analysis

independent variables

Pol γA WT
Exo-deficient [D198A/E200A (exo-)]
Pol γA R853A
Pol γA R853Q
Pol γA R853W
Pol γA R853A (exo-)
Pol γB WT
Pol γB-ΔI4 (deletion of amino acids 136 to 182)

dependent variables

Protein purity (estimated to be >95% per SDS–polyacrylamide gel electrophoresis analysis)

control variables

Expression system (insect cells Sf9 for Pol γA mutants, E. coli Rosetta BL21 DE3 for Pol γB mutants)
Purification methods (TALON, Superdex 200, Nickel-NTA, Mono S column chromatography)
Mixing ratio of Pol γA and Pol γB (1:2 molar ratio)

controls

Positive control: Pol γA WT and Pol γB WT
Negative control: Not explicitly mentioned

Annotations

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