Hiload 16 60 superdex 75 prep grade column

Purification of His-tagged proteins (His-RppH, His-DapF, His-RppH(E56&57A), His-DapF(C73&217A) and His-EIIA^Ntr) was performed as previously described with some modifications (7 (link)). E. coli GI698 strains harboring pRE1-based expression vectors were grown and protein expression was induced as described previously (25 (link)). The pellet of cells overexpressing each His-tagged protein was resuspended in binding buffer (50-mM Tris·HCl, pH 8.0, containing 300-mM NaCl) and then passed two times through a French pressure cell at 10 000 p.s.i. The lysate was cleared of cell debris by centrifugation at 100 000 x g for 90 min. The soluble fraction was loaded onto a BD TALON^TM metal affinity resin (BD Biosciences Clontech) and bound proteins were eluted with binding buffer containing 200 mM imidazole. The fractions containing His-tagged protein were pooled and concentrated in an Amicon Ultracel-3K centrifugal filter (Millipore). To remove imidazole and to purify the protein to homogeneity (>98% pure), the concentrated pool was chromatographed on a HiLoad 16/60 Superdex 75 prepgrade column (GE Healthcare Life Sciences) equilibrated with 50 mM Tris-HCl (pH 8.0) containing 100 mM NaCl. The fractions containing the protein were pooled and concentrated as described above. The purified protein was stored at –80°C until use.

Recombinant GST-CDK5, GST-CIV and 6His-p25 were expressed in E. coli following induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 5 h at 25°C (GST-CDK5), or overnight at 20°C (GST-CIV) and purified by chromatography first by affinity GST-Trap HP columns (GE Healthcare) followed by size exclusion chromatography on Hiload 16/60 Superdex 75 prepgrade columns (GE Healthcare) equilibrated in TBS buffer (50 mM TRIS-HCl, pH 7.4, 150 mM NaCl), respectively. Recombinant Tau was expressed in E. coli following induction with 0.5 mM IPTG for 3 h at 37°C. The soluble-protein fraction was incubated for 15 min at 75°C and centrifuged to pellet precipitated proteins. The supernatant was incubated with 60% ammonium sulfate overnight. After centrifugation, the pellet was resuspended in TRIS buffer (50 mM TRIS-HCl, pH 7.4, 150 mM NaCl) and purified by FPLC on a HiLoad 16/60 Superdex 75 prep-grade column (GE Healthcare) equilibrated in TRIS buffer.

These scaffold proteins were expressed with a thrombin-cleavable N-terminal His₆ tag in BL21 (DE3) E. coli induced with 0.5 mM isopropyl -D-1-thiogalactopyranoside at OD₆₀₀ of 0.6 at 16 °C for 16 h. Cells were harvested, pelleted and stored at −20 °C. Cells were lysed using an Avestin EmulsiFlex C3 cell crusher and centrifuged at 4 °C, 13,000g for 40 min to remove insoluble cell debris. Scaffold proteins were purified from the soluble fraction by affinity chromatography over Talon Superflow resin (Takara Clontech), which was equilibrated with 20 mM Tris-HCl, 150 mM NaCl, and 5 mM imidazole at pH 7.4 and were eluted with 400 mM imidazole in 20 mM Tris-HCl, 150 mM NaCl at pH 7.4. The His₆ fusion partner was cleaved by thrombin enzyme (Sigma-Aldrich). Scaffold proteins were further purified using a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare), where it elutes as a single monomeric peak in 20 mM Tris-HCl, 150 mM NaCl at pH 7.4. Collected fractions were pooled, buffer exchanged to 50 mM Imidazole, 10 mM CaCl₂ and pH 6.8 for recording NMR experiments.

BL21 (DE3) E. coli competent cells (Life Technologies, Carlsbad, CA) transformed with a pET-28a vector containing KM10 or K-11 scFv were grown in lysogeny broth at 37°C. scFv expression was induced by adding 1 mmol/L isopropyl-β-D(−)-thiogalactopyranoside (Wako) and incubated at 37°C for 5 h. After sonication for 15 min, the pellet was solubilized in 50 mmol/L Tris-HCl (pH 7.6) containing 6 mol/L guanidine-HCl and 10 mmol/L 2-Mercaptoethanol at 4°C. The solubilized scFv was purified using Ni-NTA agarose (Qiagen, Venlo, Netherlands). The purified scFv was reduced by adding 10 mmol/L 2-mercaptoethanol for 2 h at room temperature. Furthermore, the denatured 7.5 μmol/L scFv was refolded according to the method by Tsumoto et al. [18 (link)] The refolded scFv was purified by size exclusion chromatography on a HiLoad 16/60 superdex 75 prep grade column (GE Healthcare, Buckinghamshire, UK).

Polyhistidine-tagged recombinant proteins were obtained by autoinduction (56 (link)) and purified by affinity and gel filtration chromatography. Briefly, BL21(DE3)(pLysS) cells harboring the LIC vectors were grown in ZYP-5052 (1 liter) overnight at 28°C at 250 rpm. Cells were harvested by centrifugation at 18,600 × g, resuspended in a mixture of 25 mM Tris-HCl (pH 7), 150 mM NaCl, 0.5% Triton X-100, protease inhibitor cocktail (Sigma), and DNase I (Roche), and lysed by sonication (Misonix, Inc., XL-2000; QSonica). Cell debris was removed by centrifugation, and histidine-tagged proteins were purified using Talon metal affinity resin (Clontech) preequilibrated with buffer A (25 mM HEPES [pH 7], 150 mM NaCl). Proteins bound to the resin were washed extensively with buffer B (25 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole) and eluted with buffer C (25 mM Tris-HCl [pH 7], 150 mM NaCl, 250 mM imidazole). Fractions containing the eluted protein were pooled and dialyzed overnight in buffer A and further purified by gel filtration chromatography (Äkta, GE Healthcare) using a HiLoad 16/60 Superdex 75 prep-grade column (GE Healthcare) preequilibrated in buffer A.

His₆-SUMO-FtsN(51-266) was overproduced in E. coli AI060 and isolated as described for His₆-SUMO-DipM(459-609), with the addition of a size exclusion chromatography step to improve the purity of the preparation. After removal of the SUMO tag, fractions from the second Ni-NTA affinity purification step that were highly enriched in FtsN(51-266) were concentrated and applied to a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare, USA) equilibrated with buffer B6. Fractions containing the protein at high purity were pooled, concentrated, aliquoted, snap-frozen and stored at −80 °C.

The scaffold proteins were expressed in BL21 (DE3) cells and purified as described for the RXFP1 scaffold protein previously^{27 (link)}. Briefly, after 16 h of expression at 16 °C via isopropyl –D-1-thiogalactopyranoside (IPTG) induction in LB medium after cells reached an OD₆₀₀ of 0.6, cells were harvested, pelleted and purified by affinity chromatography over Talon Superflow resin (Takara Clontech). The His₆ affinity tag was removed by thrombin cleavage and the proteins further purified with a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) in 20 mM Tris HCl (pH 7.4), 150 mM NaCl. Purity and molecular mass was assessed by polyacrylamide gel electrophoresis and mass spectrometry.