Hiload 26 60 superdex 200 pg column

The cloning, expression and purification of wild type R17 and its variants has been described in Lubman et al. (Lubman et al., 2014 (link)). For crystallization, we employed an alternate version of the published protocol developed to minimize the amount of N-linked carbohydrate (Chang et al., 2007 (link)). This involved expression of both R17 and R17^GAG2 mutant in medium containing 1mM of glycosylation processing inhibitor kifunensine. The culture medium was collected 10 days after transfection and was purified using Ni-Agarose beads (Qiagen, Valencia, CA). The eluted protein was buffer exchanged into 50mM Hepes pH7.5, 600mM NaCl and incubated at room temperature overnight with Endoglycosidase Hf (3000U of EndoH for 1μg of protein) (New England Biolabs). The digested material passed over an amylose column to remove the EndoHf/maltose-binding protein fusion, followed by size exclusion chromatography (SEC) on a HiLoaD 26/60 Superdex 200pg column (G.E. Healthcare). For purification of the wild type R17, the NaCl concentration was maintained at 600mM throughout purification and crystallization. For the R17^GAG2 variant, the NaCl concentration was maintained at 150mM for subsequent co-purification with CCL3 (see below).

The mouse CCL3 variant (D26A) was produced in E.Coli and harbors the mutation D26A. The purification of the R17^GAG2 variant is described in the above section. The two proteins were mixed in 1:5 molar ratio of R17 to CCL3. The 1:1 complex was purified using size exclusion chromatography (SEC) on the HiLoaD 26/60 Superdex 200pg column (G.E. Healthcare). Crystals of the R17^GAG1/CCL3 complex at 60mg/ml were grown using 22% PEG3350 and 0.4M Mg Nitrate. Crystals of the complex belong to the I222 space group, with two molecules of R17^GAG2 and two molecules of CCL3 (D26A) in the asymmetric unit. Native data was collected at the ALS beamline 4.2.2 (Lawrence Berkeley Laboratories) at a wavelength of 1Å at 100K with a CCD detector. The structure of R17 alone and human CCL3 (PDB: 2X69) was used to solve the structure of the complex by molecular replacement using Phaser within Phenix (Adams et al., 2011 (link)). The final model has an R-value of 21.52% and R_free of 27.40%. The refined atomic model of R17^GAG2/CCL3(D26A) comprises residues 18–400 Chain A/Chain B of R17 and residues 7–68 Chain D/Chain E of CCL3 along with 2 N-linked glycosylation sites for each of R17 chain. Due to poor electron density, residues 249–254 of Chain A and 247–254 of Chain B were not included in the final model. All of the structural analysis described in the paper was done on AD complex.

A HiLoad 26/60 Superdex 200 pg column (GE Healthcare) was equilibrated in 200 mM Na-phosphate pH 7.6, 10% glycerol and calibrated using commercially available protein standards (Sigma MWGF1000-1KT) at a 1.0 mL/min flow rate. K_av = (V_e-V_o)/(V_t-V_o), where V_e, V_o, and V_t are apparent elution volume, void volume, and total volume, respectively. V_o and V_t were determined with dextran blue 2000 and adenine, respectively. Protein samples (500 μL, 10-30 mg/mL) were loaded, and eluted at a flow rate of 1 mL/min.