Protein purification

3.3.2.1 NAC and derivatives

Codon optimized EGD1 with a C-terminal TEV-cleavable His6 tag and wildtype EGD2 was cloned into

pET-Duet-1. Co-expression of the NAC-subunits in E.coli ER2566 of the subunits was performed in 2.5 l LB medium according to section 3.2.3. For Cystein derivatives, all Buffers were supplemented with 2 mM DTT. The cells were lysed and the cleared lysate incubated with 6 ml pre equilibrated Ni-NTA for 1 hour at 4°C. The column was washed five times with 7 column volume (CV) buffer A plus 5 mM imidazole and the complex eluted in 6 steps of one CV buffer A plus 500 mM imidazole each. The peak fractions were pooled and together with 300 µl TEV protease (7 A280/ml) dialyzed against 4 l of

buffer B at 4°C. One third of the dialysate was used for further purification. Anion exchange column:

The dialysed sample was supplemented with 1 mM ATP to activate the ATP cycle of chaperones. Thereby chaperones bound to the intrinsically disorded region are released and NAC could be purified to homogeneity. The sample was incubated for 15 minutes on ice and then loaded on a Poros-HQ20 column (4.6 ml, life technologies) using an äkta purifier system. Unbound sample was washed out with two CV buffer HQ without salt and elution was performed via a gradient from 0-1 M KCl in buffer HQ. The gradient consisted of two CVs from 0 to 5 mM KCl in buffer B followed by 14 CVs from 5 to 1,000 mM KCl in buffer HQ. NAC eluted between 335-550 mM KCl The fractions containing NAC (335-550 mM KCl) were pooled and diluted to 100 mM KCl in buffer HQ.

42 Cation exchange column:

The pooled fraction were then loaded on a Poros-HS20 column (4.6 ml), unbound sample was washed out with two CVs buffer HQ without salt and elution was performed via a gradient from 0-1 M KCl in buffer HQ. The gradient consisted of two CVs from 0 to 5 mM KCl in buffer B followed by 35 CVs from 5 to 1000 mM KCl. NAC eluted between 270-420 mM KCl. The peak fractions (270-420 mM KCl) were pooled.

Gel permeation chromatography column:

The pooled fractions were concentrated by filtration to 0.6 ml (Amicon ultra 10 k, Millipore) and loaded onto a S75 16/60 gel permeation chromatography column (GE healthcare, Buffer B). The fractions containing pure NAC were pooled, the protein concentration determined using the BCA assay and the sample was flash frozen and stored at -80 °C until further use.

When Cystein derivatives were purified, all buffers contained 2 mM DTT. Buffer A 50 mM Tris-Cl, pH 8.0 500 mM NaCl Buffer B 50 mM Tris-Cl, pH 8.0 50 mM NaCl 1 mM EDTA Buffer HQ/HS 20 mM Hepes, pH 7.4 0/1000 mM KOAc 3.3.2.2 MBP-Fusion proteins

MBP-Fusion proteins were cloned in either pMAL-c2 or pET28a plasmids. Expression for all derivatives was best in E. coli Rosetta (DE3) and expression of a 2 liter culture was done according to section 3.2.3 (p. 38) with the modification that the LB medium was supplemented with 1 % glucose to repress expression of amylase. The cells were lysed and clear by ultracentrifugation (see section 3.3.1) and the lysate incubated with 2 ml amylose resin (equilibrated in MBP-200 buffer) per liter starting culture. The flow-through was reloaded on the resin once. Unbound sample was washed away with one CV MBP-200 buffer. The column was washed 7 times with two CVs each of MBP buffer with increasing NaCl concentration (200 mM, 300 mM, 500 mM, 700 mM, 800 mM, 1,000 mM, 1500 mM). Before elution, the column was equilibrated to low salt by washing with two CVs of MBP-200. The MBP Fusion proteins were eluted in 4 steps using two CVs of buffer MBP-200 plus 10 mM maltose each. Protein concentration of the MBP fusion proteins was determined by measuring

43 A280 (see also section 3.4.1). The proteins were concentrated, aliquoted, flash frozen and stored at -

80 °C until further use. MBP-X buffer

20 mM Hepes, pH 7.4 1 mM EDTA

X mM NaCl

3.3.2.3 Soluble SRP-receptor

The α subunit of S. cerevisiae SRP-receptor in the pET28a vector and a truncated version of the β subunit (Δ31-244) in frame with a PreScission-protease cleavable N-terminal His6-tag in pET21a were

provided by Marko Gartmann (AG Beckmann, Gene center LMU Munich). The plasmids were co- transformed into E. coli ER2566 and expression in 12 liter LB medium plus Ampicillin and Kanamycin was done according to section 3.2.3 (p. 38). Purification based on the His6-tag was done according to

section 3.3.2.1 with 7 ml Ni-NTA per 12 liters medium. Dialysis was done in the presence of 40 Units of PreScission-protease against SR-Buffer to cleave the N-terminal His6 tag.

SR buffer

20 mM Tris-Cl, pH 7.5 150 mM NaCl 0.5 mM EDTA

Cation exchange column:

The dialyzed sample was loaded onto a Poros HS20 column (4.6 ml, life technologies). A gradient from 0-1 M KOAc in HQ buffer over 20 CVs was applied to elute the complex. The peak fractions (240-500 mM KOAc) were pooled, concentrated by filtration (Amicon ultra 10 k, Millipore), flash frozen and stored at -80 °C until further use. The concentration was determine using the BCA-assay. Buffer HQ/HS

20 mM Hepes, pH 7.4 0/1000 mM KOAc

3.3.2.4 TF

Plasmids coding for wild type TF or the single cystein mutant R150C in pPROEX-Hta were kindly provided by Ulrich Hartl, MPI Biochemie in Martinsried (Kaiser et al., 2006). The plasmid was transformed into E. coli ER2566 and expression in 2 Liter LB-medium plus Ampicillin was performed according section 3.2.3 (p. 38). The cells were lysed. For Cystein derivatives, all Buffers were supplemented with 2 mM DTT.

44 Purification based on the His-tag was done according section 3.3.2.1 with 1 ml Ni-NTA resin per 2 liters medium. To cleave the His6-tag, the elution of the Ni-NTA column was incubated at 34°C for 2h

in the presence of 40 µl TEV-protease (7,8 A280/ml). TF was then dialysed over night against 50 mM

Tris, 50 mM NaCl. Anion exchange column:

The dialysed sample was loaded onto a Poros HQ20 column (4.6 ml, life technologies). A two step gradient from 0-1M KOAc in 20 mM Hepes, pH 7.4 over 17 CVs was applied to elute the complex (0- 50 mM KOAc, 2 CV and 50-1,000 mM KOAc, 15 CV). TF eluted at 170-280 mM KOAc. The peak fractions (170-280 mM KOAc) were pooled, concentrated by filtration (Amicon ultra 10 k, Millipore), flash frozen and stored at -80 °C until further use.

Gel filtration column:

The pooled fractions were concentrated to 0.6 ml (Amicon ultra 10 kDa cut off, Millipore) and loaded onto a S200 26/60 gel permeation chromatography column in 20 mM Hepes, 100 mM KOAc, pH 7.4. The fractions containing only full length TF (elution volume 78-90 ml) were pooled, the protein concentration determined using the absorption at A280 and the sample was flash frozen and stored

at -80 °C until further use.