Purification of Human TFIIH p62 and RPB6

Unlabeled or ¹³C/¹⁵N-labeled human TFIIH p62 PH-D (residues 1–108) and unlabeled or ¹³C/¹⁵N-labeled human RPB6 (residues 1–127) were prepared as previously described (5 (link)). In brief, p62 or RPB6 was expressed as a hexa-histidine-tagged product in a pET15b vector (Merck Millipore) in Escherichia coli BL21 (DE3) Gold (Agilent Technologies). The lysed supernatant was loaded onto a Ni-nitrilotriacetic acid (NTA) column (QIAGEN), and the eluate was digested with thrombin to remove the histidine tag. After concentration with an Amicon Ultra device (Merck Millipore), the sample was purified on a Superdex75 column (GE Healthcare).

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Okuda M., Suwa T., Suzuki H., Yamaguchi Y, & Nishimura Y. (2021). Three human RNA polymerases interact with TFIIH via a common RPB6 subunit. Nucleic Acids Research, 50(1), 1-16.

Publication 2021

pET15b vector Escherichia coli BL21 (DE3) Gold Ni-nitrilotriacetic acid (NTA) column Amicon Ultra device Superdex75 column

Device Escherichia coli Gold Hexa Histidine Human Nitrilotriacetic acid Thrombin Vector

Corresponding Organization : Hiroshima University

Other organizations : Tokyo Institute of Technology

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Variable analysis

independent variables

Presence or absence of 13C/15N labeling for p62 PH-D (residues 1–108) and RPB6 (residues 1–127)

dependent variables

Not explicitly mentioned

control variables

Expression of p62 or RPB6 as a hexa-histidine-tagged product in a pET15b vector in Escherichia coli BL21 (DE3) Gold
Purification of p62 or RPB6 using Ni-nitrilotriacetic acid (NTA) column, thrombin digestion to remove the histidine tag, and Superdex75 column

controls

Unlabeled p62 PH-D (residues 1–108) and RPB6 (residues 1–127) as negative controls
13C/15N-labeled p62 PH-D (residues 1–108) and RPB6 (residues 1–127) as positive controls

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