Figure 1.
Schematic description of the plasmids used in the study. CMV, cytomegalovirus promoter; Ig-κ, Ig-κ chain leader sequence; filled black boxes, GGGS-spacer. (A) The expression plasmid for the extracellular domain of FcRn: αFcRnECD, the extracellular domain of FcRn; FLAG, the FLAG-tag; bsd, blasticidin resistance gene. (B) The expression plasmid for human β2m: His6, hexa-histidine tag; neo, G418 sulfate resistance gene. (C) The expression plasmid for full-length FcRn as a fusion to enhanced green fluorescent protein (eGFP) including the endogenous signal peptide of FcRn α-chain (SP).
Figure 2.
Representative chromatogram of the purification of FcRnECD.
Human FcRnECD with retained IgG binding was purified in a single affinity chromatography step on immobilized human IgG. The x-axis corresponds to the volume after sample injection on the column. The y-axis corresponds to measured absorbance. The dotted line corresponds to the start of elution where the pH is changed from 5.8 to 8.1.
Figure 3.
SDS-PAGE and Western blot analysis.
Eluted fractions (1-3) from the representative purification of FcRnECD were analyzed by SDS-PAGE. The gel was stained with GelCode Blue Stain Reagent or was silver stained and showed essentially only two bands with a molecular mass of 36 and 12 kDa. WB: western blotting of pooled fractions using antibodies specific for FcRn and human β2m, identifying the protein bands as αFcRnECD and β2m.
Figure 4.
(A) Representative spectrum of FcRnECD determined by circular dichroism spectroscopy. (B) The pH-dependent interaction between FcRnECD and IgG was investigated by ELISA. Immobilized FcRnECD was probed with human IgG at different pH values. Each data point is an average of three experiments with standard deviation. (C) The functionality of the FLAG-tag and the His6-tag was investigated by ELISA. Immobilized IgG was probed with FcRnECD followed by an α-FLAG antibody or an α-His6 antibody. In the control experiments FcRnECD was omitted. Each data point is an average of three experiments with standard deviation. (D) The tendency to precipitate or be degraded during storage was investigated by exposing FcRnECD to 8°C (Lane 1), -20°C (Lane 2), repeated cycles of freezing and thawing (Lane 3) or 37°C (Lane 4) during 48 h.
Figure 5.
SPR analysis of ligands binding to FcRnECD.
Overlay of the sensorgram obtained after injection of 100 nM of Bevacizumab (A), 10 μM human SA (B) or 1 μM 1-D1K (C) at pH 6.0 or pH 7.4 over immobilized FcRnECD. (D) Serial dilution of Bevacizumab injected over immobilized FcRnECD at pH 6.0. The sensorgram is an overlay of duplicate injections of each concentration and fitted curves obtained after evaluation. (E) Serial dilution of human SA injected over immobilized FcRnECD at pH 6.0. The sensorgram is an overlay of duplicate injections of each concentration.
Figure 6.
Image of a representative cell expressing human FcRn-eGFP.
The cells were stained with DAPI (blue) and eGFP was colored green.
Figure 7.
pH-dependent binding of human IgG or HSA to human FcRn-eGFP.
HeLa cells over-expressing FcRn-eGFP was probed with human IgG or HSA. The eGFP fluorescence intensity of the cells was investigated (A, B). Cells were stained with human IgG labeled with Alexa647 (C, D) or HSA labeled with Alexa647 (E, F). Staining was performed at pH 6.0 (A, C, E) or pH 8.0 (B, D, F).