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A rapid purification method for human RNA polymerase II by two-step affinity chromatography.
Jun Hasegawa1, Masaki Endou, Takashi Narita
1Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501.
Journal of Biochemistry
|May 23, 2003
Summary
Researchers developed a faster, more effective two-step purification method to isolate active RNA polymerase II (RNAPII) complexes. This improved technique yields highly pure, functional RNAPII for in vitro transcription studies.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Transcription mechanisms are studied using in vitro systems requiring purified transcription factors.
- RNA polymerase II (RNAPII), a key enzyme, is difficult to purify due to its complex structure of at least 12 subunits.
Purpose of the Study:
- To develop an improved method for preparing highly pure and functional RNA polymerase II (RNAPII) complexes.
- To enhance the efficiency and purity of RNAPII purification for in vitro transcription studies.
Main Methods:
- Transfection of HeLa cells with a plasmid encoding a double FLAG-histidine tagged RPB3 subunit.
- Immunoaffinity purification followed by Co(2+) affinity chromatography to isolate RNAPII complexes.
Main Results:
- A high-yielding clone was established, producing RNAPII complexes containing all core subunits, including the tagged RPB3.
- The purified RNAPII complexes were demonstrated to be active and functional in basal and activated transcription assays.
- The two-step purification method achieved 92% purity, significantly higher than conventional methods (40%) and was less time-consuming.
Conclusions:
- The described two-step affinity purification method is an uncomplicated and effective approach for preparing active and functional RNAPII.
- This method significantly improves the purity and reduces the time required for RNAPII preparation compared to conventional procedures.