FIGURE 5.

Introduction of Ser¹³ into CXCL11. A, alignment of mature hCXCL10, hCXCL9, and hCXCL11 chemokine amino acid sequences. Black shaded letters indicate conserved residues according to BLOSUM 62 substitution matrix. Ser¹³ is represented in green, and Pro¹⁴ is indicated in pink. B, specific binding of E7, J9, P8, F13, C1, and J5 dual-specific scFvs to human CXCL11, its mutant, and human CXCL10 was assessed in an ELISA. The indicated NusA-fusion chemokines were captured and then incubated with scFvs. Coating was controlled using specific anti-NusA mAb, and NusA protein was also added to the assay as negative control (data not shown). Results are expressed as mean ± S.D. of duplicates. hum11, human CXCL11; hum10, human CXCL10. C, SDS-PAGE analysis of affinity-purified CXCL11 mutant and wild type, CXCL10, and NusA proteins. The proteins were denatured under reducing conditions and stained with Coomassie Blue. M, Seeblue Plus SDS molecular weight marker (Invitrogen); hum11, human CXCL11; hum10, human CXCL10. D, superimposition of the ribbon representations of monomeric hCXCL10 (dark blue) and hCXCL11 (light blue). The critical amino acid side chains at positions 13 and 14 (Ser¹³, G13S, and Pro¹⁴), and cysteine side chains are represented as sticks in green, orange, pink, and yellow, respectively.