ADP-ribosylation factor-like protein 15

Full length ARL15 gene (accession: NP_061960) was synthesized by Bio Basic Inc. (Markham, Canada) in the propagation vector pUC57. It was subsequently amplified and cloned in pET28a expression vector containing an N-terminal His tag. Since the full length ARL15 protein posed challenges to its functional characterization, due to aggregation and facile proteolysis, an N-terminal truncated mutant, N∆28 ARL15, where 28 residues were deleted at the N-terminus, was also cloned in pET28a in the same way as the full-length protein. Constructs made were confirmed by sequencing. The pET28a–ARL15 plasmid containing the gene of interest was transformed into E. coli BL21 (DE3)* cells (New England Biolabs, Ipswich, MA, USA) and plated on LB agar supplemented with 50 μg/mL of kanamycin (Bio Basic Inc., Markham, Canada). Single colony was inoculated in 10 mL of Luria Broth media for primary culture, followed by overnight incubation at 37°C at 180 rpm. This primary culture (A600= ~ 1) was used to inoculate one litre LB media (secondary culture). The secondary culture was incubated at 37°C at 180 rpm of shaking until its optical density at 600 nm reached a value of 0.8 (exponential phase). The secondary culture was induced with 0.5 mM IPTG (Bio Basic Inc., Markham, Canada) and incubated at 25°C with 180 rpm of shaking for 14 hrs. The cells were harvested at 4000xg at 4°C for 30 min. The pellet was suspended in 20 mM Tris, 500 mM NaCl, 1 mM PMSF and 10% glycerol (Thermo Fisher Scientific, Inc., MA, USA) at pH 8. Protease inhibitor cocktail (Sigma-Aldrich, St. Louis MO, USA) was also added and the suspended pellet was subjected to sonication. After sonication, the suspension was centrifuged at 9000xg for 45 min, pellet discarded and the supernatant was loaded on a column containing Ni-NTA affinity chromatography resin (HIS-Select HF Nickel Affinity Gel; Sigma-Aldrich, St. Louis, MO, USA), pre-equilibrated with 20mM Tris, 500 mM NaCl, 1 mM PMSF and 10% glycerol, pH 8. Purification was achieved using Ni-NTA immobilized metal ion affinity chromatography. Lysate was bound to the column twice over followed by excessive washing to get rid of non-specifically bound proteins. The protein of interest was eluted with a step-wise gradient of 20-150 mM imidazole (Sigma-Aldrich, St. Louis, MO, USA) in the same buffer as mentioned above. All the fractions were subjected to SDS-PAGE and fractions containing the protein of interest were pooled. The eluted protein was then concentrated and subjected to size exclusion chromatography (Superdex 75 16/600 GL;GE Healthcare Life Sciences, Buckinghamshire, UK) column pre-equilibrated with the final storage buffer consisting of 20 mM Tris, 100 mM NaCl, 1 mM PMSF, 10% glycerol and 1 mM DTT (Thermo Fisher Scientific, Inc., MA, USA) (pH 8). The fractions containing the protein of interest were then stored at 4°C.