FIGURE 2.
Generation by site-directed mutagenesis and soluble expression of CXCL10 and CXCL9 mutants in E. coli. A, schematic of chemokine construct. Recombinant chemokines were generated by site-directed mutagenesis, PCR assembly, enzymatic digestion, and cloning into pET43 vector for E. coli expression. All resulting chemokines were expressed as NusA fusions proteins and contained both N- and C-terminal histidine tags for purification purposes. His6, 6-histidine tag; Xa, cleavage factor; Avi, AviTagTM (avidity). X indicates an enzymatic restriction site. B and C, SDS-PAGE analysis of affinity-purified CXCL10 and CXCL9 mutants, respectively. Affinity-purified CXCL10 (B) and CXCL9 (C) mutants and NusA protein were denatured under reducing conditions and stained with Coomassie Blue. M, Seeblue Plus molecular weight marker (Invitrogen); hum10, human CXCL10; rab10, rabbit CXCL10; hum9, human CXCL9; cyn9, cynomolgus CXCL9.