Ni nta

MUT was expressed in E. coli and purified as previously described [Froese et al., 2010b] with minor modifications. For small‐scale purification, cells were grown in a total of 50 ml, induced with 0.1 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 18°C overnight, harvested by centrifugation at 4,000g, lysed by sonication, and purified by affinity (Ni‐NTA; Qiagen, Venlo, The Netherlands) chromatography. Where applicable, chemical chaperones were added concurrently with the IPTG. Samples from total cell lysate (1 μl of 2 ml total) (“L”), including all cellular proteins both soluble and insoluble, and affinity eluants (15 μl of 250 μl total) (“E”), including those soluble proteins eluted from the nickel affinity column, were analyzed by SDS‐PAGE and stained with Coomassie blue (Expedeon, San Diego, CA, USA). For large‐scale purification, cells were grown in a total of 6 l, harvested by centrifugation at 5,000g, lysed with an Emulsiflex C3 homogenizer, and purified by affinity (Ni‐NTA, Qiagen), size‐exclusion (Superdex 200; GE Healthcare, Little Chalfont, UK), cleavage with His‐tagged TEV protease and reverse‐affinity chromatography (Ni‐NTA). Purification buffers are detailed in the webpage http://www.thesgc.org/structures/details?pdbid=2XIQ.

The recombinant gpT5.026 protein was labeled with an isotope ³²P using the catalytic subunit of protein kinase A (Sigma), as described [20 (link),21 (link),22 (link)], followed by a clean-up on Ni–NTA (Qiagen). Far-Western blotting was performed as previously described [21 (link),23 (link)]. Briefly, 1 μg of purified proteins (gpT5.026 or gp2) were incubated with 20 units of protein kinase A in PKA buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 30 mM DTT, 10 mM MgCl₂) in the presence of 0.4 mCi of γ-[³²P] ATP at 30 °C for 1 h, followed by a clean-up on Ni-NTA (Qiagen). 1 μg of RNAP core enzymes or individual RNAP subunits (α, β, β’, and σ⁷⁰) were applied as small drops on a Hybond ECL membrane and annealed for 3 min at 50 °C. Then, membranes were blocked in PROB buffer (20 mM 1,4-Piperazinediethanesulfonic acid (PIPES) (pH 7.4), 200 mM KCl, 1 mM DTT, 2 mM MgCl₂, 10% glycerol, 0.5% Tween-20, 1% nonfat dried milk) for 2 h at room temperature. Afterwards, each piece of membrane was enveloped in a parafilm sack containing 150 μL of radiolabeled proteins solutions with/without added non-labeled proteins (depending on the design of experiment) in PROB buffer and incubated for 2 h at room temperature. Next, membranes were washed three times with 1 mL of PROB buffer and dried at room temperature for 15 min. Results were revealed using a PhosphorImager (Molecular Dynamics).

The cDNAs of Apg2 (HSPH2), Hsc70 (HSPA8), and DNAJB1 were obtained from Addgene and cloned into pE-SUMO vector (LifeSensors, Malvern, USA). The mutants Apg2ΔAS and Apg2ΔC, carrying a deletion from residue 504 to 569 and 702 to 840, respectively, were cloned by fusing two PCR fragments corresponding to the upstream and downstream sequences of these protein segments [88 (link)] and verified by sequencing. Recombinant proteins containing a tag with 6 histidines and SUMO fused to the N-terminus were expressed in BL21 Rosetta cells and purified with a first step of affinity chromatography using NiNTA (Qiagen, Hilden, Germany) columns, followed by treatment with His-Ulp1 to cleave the tag, and a final NiNTA column in which the pure protein eluted in the unbound fraction [89 (link)].