Recombinant protein purification protocols

Purification protocols for all recombinant proteins used in this study are listed below. All buffers are listed in Table 17.

Med8C/18/20 and Med18/20 (Sc)

The cleared lysate from two liters of culture was gradually precipitated by slowly adding saturated (NH4)2SO4 solution to a final concentration of 30%. Afterwards, suspension was stirred on ice for 30 min and centrifuged (15000 rpm / 30 min / 4°C / Sorvall SS34 rotor). Supernatant was discarded and the pellet resuspended in 8 ml of buffer A. Buffer B was added to a final conductivity of 10-15 mS/cm. The proteins were further purified by anion exchange chromatography using a MonoQ 10/100 column (GE Healthcare). The column was equilibrated with buffer B containing 100 mM NaCl and the complex was eluted with a linear gradient of 20 CVs from 100 mM to 500 mM NaCl in buffer B. Subsequently, the sample was applied to a Superdex 200 size exclusion column (GE Healthcare) equilibrated with buffer A. The protein was concentrated to approximately1 mg/ml, flash frozen in small aliquots in liquid N2 and stored at -80°C.

Med8C/18 (Sp)

The cleared lysate from three liters of culture was loaded onto a 2 ml Ni-NTA column (Qiagen) equilibrated with buffer A. The column was washed with 10 CV of buffer A containing 10 mM imidazole and with 10 CV of buffer A containing 20 mM imidazole. The complex was eluted with buffer A containing 300 mM imidazole. Buffer B was added to a final conductivity of 10-15 mS/cm. The proteins were further purified by anion exchange chromatography using a MonoQ 10/100 column (GE Healthcare). The

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column was equilibrated with buffer B containing 100 mM NaCl and the complex was eluted with a linear gradient of 10 CVs from 100 mM to 1 M NaCl in buffer B. After concentration the sample was applied to a Superose-12 size exclusion column (GE Healthcare) equilibrated with buffer A. The protein was concentrated to approximately40 mg/ml for crystallization.

Med11/22 (Sc)

The cleared lysate from three liters of culture was loaded onto a 2 ml Ni-NTA column (Qiagen) equilibrated with buffer A. The column was washed with 10 CV of buffer A containing 10 mM imidazole and with 10 CV of buffer A containing 20 mM imidazole. The complex was eluted with buffer A containing 300 mM imidazole followed by overnight cleavage with thrombin while dialyzing against buffer B. The proteins were further purified by anion exchange chromatography using a HiTrap Q HP column (GE Healthcare). The column was equilibrated with buffer B and the complex was eluted with a linear gradient of 25 CVs from 50 mM to 500 mM NaCl in buffer B. Subsequently, the sample was applied to a HiLoad Superdex-75 pg 26/60 size exclusion column (GE Healthcare) equilibrated with buffer A.

Med25-ACID (Hs)

The cleared lysate from four liters of culture was loaded twice onto a 2 ml Ni-NTA column (Qiagen) equilibrated with buffer A. The column was washed with 10 CV of buffer B containing 1 M NaCl, 10 CV of buffer A and 10 CV of buffer A containing 10 mM imidazole. The protein was eluted with 10 CV of buffer A containing 200 mM imidazole, subsequently diluted with 10 CV of buffer B containing 50 mM NaCl and further purified by cation exchange chromatography using a MonoS column (GE Healthcare). The column was equilibrated with buffer B and the complex was eluted with a linear gradient of 10 CVs from 0 mM to 1 M NaCl in buffer B. Subsequently, the sample was applied to a Superose 6 size exclusion column (GE Healthcare) equilibrated with buffer C. The protein was concentrated to approximately5 mg/ml, flash frozen in small aliquots in liquid N2 and stored at -80°C.

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Med7N/31 (Sc)

The cleared lysate from 2 liters of culture was loaded onto a 3 ml Ni-NTA column (Qiagen) equilibrated with buffer A. The column was washed with 20 CV of buffer A containing 20 mM imidazole. The complex was eluted with buffer A containing 200 mM imidazole.

followed by overnight cleavage with thrombin. The proteins were further purified by anion exchange chromatography using a MonoQ 10/100 column (GE Healthcare). The column was equilibrated with buffer B and the complex was eluted with a linear gradient of 20 CVs from 100 mM to 1 M NaCl in buffer B. After concentration, the sample was applied to a HiLoad Superdex-200 size exclusion column (GE Healthcare) equilibrated with buffer C.

TFIIB (Sc)

The cleared lysate from one liter of culture was incubated in batch (20 min / 4°C).with 2 ml Ni-NTA material (Qiagen) equilibrated with buffer A containing 5 mM imidazole, 0.2% Tween and protease inhibitor cocktail. After transferring the material to a gravity flow column, it was washed with 10 CV buffer A containing 10 mM imidazole. The protein was eluted with 3 CV of buffer A containing 200 mM imidazole. The sample was diluted with buffer B to a final conductivity of 50 mSi/cm and further purified by cation exchange chromatography using a MonoS 10/100 GL column (GE Healthcare). The column was equilibrated with buffer C and the complex was eluted with a linear gradient of 15 CVs from 100 mM to 1 M NaCl in buffer C. Subsequently, the sample was applied to a Superdex 75 10/300 GL size exclusion column (GE Healthcare) equilibrated with buffer D.

Gal4-VP16

The cleared lysate from three liters of culture was loaded twice onto a 2 ml Ni-NTA column (Qiagen) equilibrated with buffer A. The column was washed with 10 CV of buffer A, 10 CV of buffer B and 5 CV of buffer B containing 20 mM imidazole. The protein was eluted with 10 CV of buffer B containing 200 mM imidazole and further purified by anion exchange chromatography using a HiTrap Q HP column (GE Healthcare). The column was equilibrated with buffer C and the complex was eluted with a linear gradient of 10 CVs from 0 mM to 700 mM NaCl in buffer C. Subsequently, the sample was applied to a Superose 12 size exclusion column (GE Healthcare)

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equilibrated with buffer D. The sample was concentrated to approximately0.5 mg/ml, flash frozen in small aliquots in liquid N2 and stored at -80°C.

Gal4-AH

The cleared lysate from three liters of culture was loaded twice onto a 2 ml Ni-NTA column (Qiagen) equilibrated with buffer A. The column was washed with 10 CV of buffer A, 10 CV of buffer B and 5 CV of buffer B containing 20 mM imidazole. The protein was eluted with 10 CV of buffer B containing 200 mM imidazole and further purified by anion exchange chromatography using a HiTrap SP column (GE Healthcare). The column was equilibrated with buffer C and the complex was eluted with a linear gradient of 10 CVs from 0 mM to 700 mM NaCl in buffer C. Subsequently, the sample was applied to a Superose 12 size exclusion column (GE Healthcare) equilibrated with buffer D. The sample was concentrated to approximately0.5 mg/ml, flash frozen in small aliquots in liquid N2 and stored at -80°C.

Gcn4

The cleared lysate from two liters of culture was loaded twice onto a 2 ml Ni-NTA column (Qiagen) equilibrated with buffer A. The column was washed three times with 10 CV of buffer A containing 20 mM imidazole. The protein was eluted with 10 CV of buffer A containing 500 mM imidazole, subsequently diluted 1:5 with buffer B and further purified by cation exchange chromatography using a HiTrap SP column (GE Healthcare). The column was equilibrated with buffer B and the complex was eluted with a linear gradient of 10 CVs from 0 mM to 1 M NaCl in buffer B. Subsequently, the sample was applied to a Superdex 200 size exclusion column (GE Healthcare) equilibrated with buffer C. The sample was concentrated to approximately0.5 mg/ml, flash frozen in small aliquots in liquid N2 and stored at -80°C.

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