Ni2 nta affinity chromatography

Expression of GST-tagged VCF1/2 proteins or of hexahistidine-tagged UBXN7 in Escherichia coli BL21(DE3)pRIL (Agilent) was induced with 1 mM IPTG during overnight growth at 18°C. GST fusion proteins were affinity-purified on a glutathione sepharose matrix (Cytiva) according to the manufacturer’s instructions. Purified GST fusion proteins were dialyzed against TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 2 mM DTT, aliquoted, flash frozen in liquid nitrogen and stored at –80°C. His₆-UBXN7 was purified by Ni^2+-NTA affinity chromatography (QIAGEN) according to the manufacturer’s instructions, followed by anion exchange chromatography on a ResourceQ column (Cytiva). Purified His₆-UBXN7 was dialyzed against 50 mM Tris-HCl, 50 mM NaCl, 2 mM DTT, pH 8.0 and flash frozen. His₆-FAF1, His₆-p47 (Allen et al., 2006 (link)) and UFD1-NPL4, full-length and truncated p97 (Fernández-Sáiz and Buchberger, 2010 (link)) were purified as previously described.

Using the scFv 10F as a template, the combinatorial mutagenesis was made by the megaprimer strategy as described above using the oligos: C1H3A107S (5′-CCA CTC GCT GCA CAA TAG G-3′) to change A107S; C1Fw3 204A/G (5′-C CGA TTC TCT GSC TCC AAG-3′) so that position 204 could be mutated to A or G, and finally C1L1 I164S (5′-CT TGT TCT GGA AGC CGC TCC-3′) for the created I164S. The changes were made stepwise and cloned directly into the expression vector pSyn1. Once the constructs ware confirmed by sequence, the proteins of the scFvs were expressed and purified as described in [26 (link)]. The scFvs were purified by Ni2+-NTA affinity chromatography (Qiagen, Hilden, Germany), and eluted with 250 mm of imidazole. Finally, scFv preparations were purified by gel filtration chromatography on a SuperdexTM 75 column (Phamacia Biotech AB, Uppsala, Sweden). The protein concentrations of the monomers (a common characteristic in this scFv family) were determined spectrophotometrically at λ = 280 nm, using the molar extinction coefficient and the molecular weight of each scFv. For scFv, 10FG2 ε = 49,765 and MW = 28,527.34.

CotB‐His₆ and CotH‐Strep‐tag proteins were overexpressed in E. coli BL21(DE3) as described above (Fernandes et al., 2015). After the induction period, cells were collected by centrifugation (at 12,000 × g, for 10 min at 4°C). Cells with CotB‐His₆ were resuspended in one tenth of Start buffer (10 mM imidazole, 20 mM phosphate, 0.5 M NaCl), containing 1 M phenylmethanesulfonyl fluoride (PMSF). Lysates were prepared by a passage through a French press (19,000 psi) and centrifuged (at 12,000 × g, for 10 min at 4°C). Since most of CotB‐His₆ is present in the insoluble fraction, Start buffer containing urea (8 M) was added to the debris to increase the solubility of CotB‐His₆. The sample was stirred for 45 min and centrifuged (at 12,000 × g, for 10 min). CotB‐His₆ was purified by Ni²⁺‐NTA affinity chromatography (Qiagen). The Ni²⁺‐NTA affinity purified protein was analyzed by 12.5% SDS–PAGE. Fractions containing CotB‐His₆ were pooled and dialyzed against Start buffer without urea. Cells with CotH‐Strep‐tag or CotH^D228Q –Strep‐tag were resuspended in one tenth of buffer W (see above), containing 1 M PMSF. Lysates were prepared by a passage through a French press (19,000 psi) and centrifuged (at 12,000 × g, for 10 min at 4°C). CotH‐Strep‐tag or CotH^D228Q–Strep‐tag were purified using Strep‐Tactin Sepharose (IBA). The affinity purified protein was analyzed by 12.5% SDS–PAGE.

Competent E. coli BL21 (DE3) pLysS bacteria were transformed with the pRSET-EhCFIm25 plasmid^{21 (link)}. The EhCFIm25 was expressed with 1 mM isopropyl beta-D-thiogalactopyranoside (IPTG) and purified by Ni²⁺-NTA affinity chromatography (QIAGEN). The identity and integrity of the histidine-tagged EhCFIm25 protein was confirmed by 10% SDS-PAGE and Western blot assays using anti-6×-His tag antibodies (Roche) at 1:10000 dilution and the ECL Plus Western blotting detection system (Amersham).

The cDNA of the armadillo repeat (ARM) domain (residues 407–751 and 407–775) of human APC were cloned into a pET28a-derived (Novagen, Madison, WI, USA) vector and overexpressed as an N-terminally His-tagged protein. The cDNA of the APC-binding fragments of human Amer1 (full A1: 280–364, core A1: 325–335, full A2: 400–531, core A2: 496–508, A3: 766–823, and A4: 365–375) were cloned into the pGEX4T1 vector (GE Healthcare, Little Chalfont, UK) to be expressed as N-terminally GST-tagged proteins. All proteins were over-expressed in the E. coli strain BL21(DE3), and purified by the Ni²⁺-NTA affinity chromatography (Qiagen, Hilden, Germany) or the GST affinity chromatography (Sigma, St Louis, MO, USA). After further purification by the Superdex200 gel filtration chromatography, the purified proteins were concentrated to 20 mg ml⁻¹.
The peptides of Amer1-A1 (residues 325–335, LTGCGDIIAEQ), -A2 (residues 496–508, PRDSYSGDALYEF), and -A4 (residues 365–375, YQGGGEEMALP) were chemically synthesized with free amine and carboxylate ends, and purified by reverse phase HPLC (Appeptide Company, Shanghai, China). The APC/Amer1-A1, APC/Amer1-A2, APC/Amer1-A4 complexes were prepared by mixing concentrated APC–ARM proteins with the Amer1-A1, -A2, or -A4 peptides, respectively, with molar ratios of 1:1.5.