Fig 1.
SDS-PAGE analysis of cell-free synthesized 059-152-Fv and 059-152-Fab.
(A) 059-152-Fv was synthesized under a series of different concentrations (0, 0.2, and 0.4 mg/ml) of DsbC, as indicated. Total (T) and soluble (S) fractions of the internal solution were analyzed by reducing SDS-PAGE. (B) Purified 059-152-Fv was analyzed by reducing and non-reducing SDS-PAGE. The yields (mg per 1 ml internal solution) of partially purified Fv are indicated under each lane of the non-reducing SDS polyacrylamide gel image. (C) 059-152-Fab was synthesized in the presence of 0, 0.2, 0.4, and 0.8 mg/ml of DsbC, as indicated. (D) Purified 059-152-Fab was analyzed by reducing and non-reducing SDS-PAGE. The yields (mg per 1 ml internal solution) of partially purified Fab are indicated under each lane of the non-reducing SDS polyacrylamide gel image. BG: cell-free synthesis without template DNA. VH: cell-free synthesis of VH without DsbC. VL: cell-free synthesis of VL without DsbC. VHCH1: cell-free synthesis of VHCH1 without DsbC. L chain: cell-free synthesis of light chain without DsbC. Gels were stained with CBB.
Table 1.
Binding kinetics of 059-152-Fv and 059-152-Fab to the EGFR-ECD.
Fig 2.
SDS-PAGE analysis of site-specific fluorescent-labeled 059-152-Fv and 059-152-Fab.
Reducing and non-reducing SDS-PAGE analysis of 059-152-Fv (A) and 059-152-Fab (B). (lane 1) 059-152-Fv, (lane 2) AzF-incorporated 059-152-Fv, (lane 3) Alexa-488 conjugated 059-152-Fv, (lane 4) 059-152-Fab, (lane 5) AzF-incorporated 059-152-Fab, and (lane 6) Alexa-488 conjugated 059-152-Fab. Fluorescent images and CBB-stained images were acquired from the same gels.
Table 2.
Data collection and refinement statistics.
Fig 3.
Crystal structure of the 059-152-Fv•EGFR-ECD complex.
(A) Ribbon representation of the 059-152-Fv•EGFR-ECD complex. VH and VL domains of 059-152-Fv are respectively colored cyan and green. External region of the EGFR is shown with domain I in yellow, domain II in orange, domain III in red, and domain IV in purple. (B, C) Close up-view of the interactions between CDR loops of 059–152 and domain III. For clarity, interactions of CDR-H and CDR-L are separately shown. CDR-H loops, CDR-L loops, and domain III are colored cyan, green, and gray, respectively. Residues that make key interactions are shown in the stick models. Hydrogen bonds are indicated by gray dotted lines.
Fig 4.
Contacting region of 059-152-Fv, cetuximab, and EGF on domain III.
(A) Ribbon representations of domain III in complex with 059-152-Fv, EGF, and cetuximab. VH, VL, EGF, and domain III are respectively shown in cyan, green, yellow, and gray. (B) Surface representations of domain III with the contact region. Surface of domain III (gray) contacting (within 4 Å) 059–152, EGF, and cetuximab are shown in purple, red, and orange, respectively. These are viewed from approximately the same orientations onto the domain III binding site. Residues on domain III that form hydrogen bonds with EGF are shown in white.
Fig 5.
Electron density map of intradomain disulfide bonds of 059-152-Fv.
Close-up view of the electron density maps (blue mesh) and stick models of 059-152-VH (A) and 059-152-VL (B) are shown. The stick models are shown with carbons colored yellow; nitrogens, blue; oxygens, red; and sulfurs, green.