A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

Abstract

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab's constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab's constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

Figure 1

Mutation site of intermolecular SS mutants. Three-dimensional structure of adalimumab Fab (protein data bank number: 4NYL). The mutated amino acids are shown as sticks.

Figure 2

SDS-PAGE analysis of purified intermolecular SS mutants. The samples were analyzed by 12% SDS-PAGE under (a) non-reduced condition and (b) reduced condition. Lane M: protein markers. Lane 1: WT, Lane 2: ΔSSWT, Lane 3: Mut1, Lane 4: Mut2, Lane 5: Mut3, Lane 6: Mut4, Lane 7: Mut5, Lane 8: Mut6, Lane 9: Mut7, Lane 10: Mut8, Lane 11: Mut9. The gels were stained with SimplyBlue safe stain (Invitrogen) and were then imaged using iBright FL1000 (Thermo Fisher Scientific).

Figure 3

The antigen-binding activity of intermolecular SS mutants. The ELISA plate was coated with TNFα, followed by addition of intermolecular SS mutants at various concentrations. Anti-human Fab conjugated with horseradish peroxidase polyclonal antibody was used for evaluating the antigen-binding activity. The absorbance was measured at 450 nm using microplate spectrometer.

Figure 4

CD spectra of intermolecular SS mutants. The intermolecular SS mutants were dialyzed against 50 mM KH₂PO₄ buffer, pH 6.5, and prepared at a concentration of 0.2 mg/mL. Then, the CD spectra were collected on a spectropolarimeter.

Figure 5

DSC measurements. Thermograms of Mut3, Mut4, Mut5, Mut6, Mut7, and Mut8 monitored from 25 and 90 °C at a scan rate of 60 °C/h. The protein solutions were prepared at concentration of 0.2 mg/mL in 50 mM KH₂PO₄ buffer, pH 6.5.

Figure 6

SDS-PAGE analysis of refolded Fab. The samples of (a) refolded WT and (b) refolded Mut1 analyzed by 12% SDS-PAGE under non-reduced condition. The gels were stained with SimplyBlue safe stain (Invitrogen) and were then imaged using iBright FL1000 (Thermo Fisher Scientific).