Recombinant CXCL12 Expression and Purification

The chemokine CXCL12 was expressed and purified as previously described^{52 (link)}. Briefly, mature chemokine sequence, preceded by an 8His tag and an enterokinase cleavage site, was cloned into a pET21-based vector and expressed in BL21(DE3)pLysS cells by IPTG induction. Cells were lysed by sonication and chemokine containing inclusion bodies were dissolved in 50 mM Tris, 6 M guanidine-HCl, 50 mM NaCl, pH 8.0. The chemokines were bound to a Ni-NTA column, washed with 50 mM MES, 6 M guanidine-HCl, 50 mM NaCl, pH 6, and eluted with 50 mM acetate, 6 M guanidine-HCl, 50 mM NaCl, pH 4. CXCL12 was refolded in 50 mM Tris, 500 mM arginine-HCl, 1 mM EDTA, 1 mM glutathione disulfide, pH 7.5 before removal of the tag by enterokinase. The cleaved material was then bound to a C18 HPLC column (Vydac) (buffer A: 0.1% trifluoroacetic acid; buffer B: 0.1% trifluoroacetic acid, 90% acetonitrile) and eluted by a linear gradient of buffer B from 33–45%. The peak was collected, lyophilized, and stored at −80°C until use.