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.
Highlights
Monoclonal antibodies are highly effective against intractable diseases, such as cancer and r heumatism[1,2,3]
To facilitate the analysis of intermolecular disulfide bond formation, mutations were made to fragment antigen binding (Fab) in which the cysteine residues to form intact C-terminal intermolecular disulfide bond (C224(H):C214(L)) in human Fab were replaced with alanine (ΔSSWT); resulting that we can detect quickly whether or not an intermolecular disulfide bond in mutant Fabs form using SDS-PAGE of respective mutant treated in the absence or presence of reductant
It was observed that mutations in the adalimumab Fab-H chain (V177C) and L chain (Q160C) could successfully enable the introduction of an intermolecular disulfide bond in the constant domain[27]
Summary
Monoclonal antibodies are highly effective against intractable diseases, such as cancer and r heumatism[1,2,3]. Intermolecular disulfide bond generally contribute to the conformational protein stability. A previous study has shown that the introduction of intermolecular disulfide bond increased the conformational stability of dimer p rotein[23,24,25,26]. Few attempts have been made to report the introduction of a novel intermolecular disulfide bond into the Fab molecule to increase protein stability (Supplementary Figures 1 and 2). An expression system of a Fab of IgG1 developed as the therapeutic antibody adalimumab, an anti-human TNF-α, was constructed in the yeast strain P. pastoris. In order to identify the further sites that facilitated the introduction of intermolecular disulfide bond within the Fab’s constant domain and to collect information on its stabilization, other presumed mutants with engineered intermolecular disulfide bond were prepared using P. pastoris
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