Fig. 7
From: A loop region of BAFF controls B cell survival and regulates recognition by different inhibitors
Structure of the BAFF–belimumab Fab complex. The Fab of belimumab was generated by papain digestion, and a 3-mer of BAFF was obtained by expression of BAFF H218A in bacteria. The complex formed by the association of BAFF 3-mer with belimumab Fab was isolated by size-exclusion chromatography and characterized by electron microscopy and by crystallography. The activity of BAFF 3-mer alone or in complex with belimumab Fab was tested on BAFFR:Fas reporter cells. a Elution profile of molecular weight standards by size-exclusion chromatography. b Elution profile of belimumab Fab. The experiment was performed twice. c Elution profile of His-BAFF H218A. The experiment was performed twice. d Elution profile of BAFF 3-mer mixed with an excess of belimumab Fab. The experiment was performed twice. For panels b, c, and d, collected fractions are indicated by the underlying red line. e Elution profile of the isolated BAFF–belimumab Fab complex. The experiment was performed twice. f 5 µg of BAFF 3-mer, 15 µg of belimumab Fab, and 20 µg of the BAFF–Fab complex were analyzed by reducing (+DTT) or non-reducing (−DTT) SDS-PAGE and Coomassie blue staining. The migration positions of BAFF and of the Fab chains are indicated on the right. The experiment was performed twice. g–k Superposition of typical electron microscopy negative stain images with the crystal structure of the BAFF–belimumab Fab complex. Note that in panels j and k, respectively one and two Fab were removed from the crystal structure. The experiment was performed once. Scale bars: 100 Å. l Biological activities of BAFF 3-mer and of BAFF–belimumab Fab complexes were measured on reporter cells. The experiment was performed three times. See also Supplementary Table 4
