I am thinking about purifying some Cas9 and was wondering if anyone had any tips/suggestions before I start doing some in-depth research.
Some specific questions:
Tagged vs untagged? (does the tag have any effect on Cas9 activity/specificity)
Best cell type/expression vector?
Expected yields?
Solubility issues?
Any advice would be appreciated.
Thanks,
John
Hi, Jonathan, I remember the crystal structure being solved for this protein last year. That's good news because a lot of protein is typically desired for crystallization so you can duplicate their work. At the very least, it's a good place to start.
Answers to your questions:
Nishimasu et al. used a his6x-GST tag and cleaved with a protease.
Cell type: Escherichia coli Rosetta 2 (DE3)
I would expected yields to be high, esp. since Cas9 is a bacterial protein. Probably 10-20mg/mL.
No solubility issues are mentioned in the paper.
From the "Experimental Procedures" section:
The S. pyogenes Cas9 D10A/C80L/C574E/H840A mutant (residues 1–1368) was expressed in Escherichia coli Rosetta 2 (DE3) (Novagen) and purified by chromatography on Ni-NTA Superflow (QIAGEN), Mono S (GE Healthcare), and HiLoad Superdex 200 16/60 (GE Healthcare) columns.
From the "Extended Experimental Procedure section":
The gene encoding full-length S. pyogenes Cas9 (residues 1–1368) was cloned between the NdeI and XhoI sites of the modified pCold-GST vector (TaKaRa), and mutations were introduced by a PCR-based method. The S. pyogenes Cas9 D10A/C80L/C574E/H840A mutant was expressed at 20°C in Escherichia coli Rosetta 2 (DE3) (Novagen), and was purified by chromatography on Ni-NTA Superflow resin (QIAGEN). The eluted protein was incubated overnight at 4°C with TEV protease to remove the His6–GST-tag, and was further purified by chromatography on Ni-NTA, Mono S (GE Healthcare) and HiLoad Superdex 200 16/60 (GE Healthcare) columns.
Article Crystal structure of Cas9 in complex with Guide RNA and target DNA
Hi, Jonathan, I remember the crystal structure being solved for this protein last year. That's good news because a lot of protein is typically desired for crystallization so you can duplicate their work. At the very least, it's a good place to start.
Answers to your questions:
Nishimasu et al. used a his6x-GST tag and cleaved with a protease.
Cell type: Escherichia coli Rosetta 2 (DE3)
I would expected yields to be high, esp. since Cas9 is a bacterial protein. Probably 10-20mg/mL.
No solubility issues are mentioned in the paper.
From the "Experimental Procedures" section:
The S. pyogenes Cas9 D10A/C80L/C574E/H840A mutant (residues 1–1368) was expressed in Escherichia coli Rosetta 2 (DE3) (Novagen) and purified by chromatography on Ni-NTA Superflow (QIAGEN), Mono S (GE Healthcare), and HiLoad Superdex 200 16/60 (GE Healthcare) columns.
From the "Extended Experimental Procedure section":
The gene encoding full-length S. pyogenes Cas9 (residues 1–1368) was cloned between the NdeI and XhoI sites of the modified pCold-GST vector (TaKaRa), and mutations were introduced by a PCR-based method. The S. pyogenes Cas9 D10A/C80L/C574E/H840A mutant was expressed at 20°C in Escherichia coli Rosetta 2 (DE3) (Novagen), and was purified by chromatography on Ni-NTA Superflow resin (QIAGEN). The eluted protein was incubated overnight at 4°C with TEV protease to remove the His6–GST-tag, and was further purified by chromatography on Ni-NTA, Mono S (GE Healthcare) and HiLoad Superdex 200 16/60 (GE Healthcare) columns.
Article Crystal structure of Cas9 in complex with Guide RNA and target DNA
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