Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.
Bottom Line: We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization.Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties.Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.
Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.Show MeSH
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Mentions: Bi-cistronic pIRES vectors containing the engineered sequences were used for transfection of CHO DG44 cells [12,14], giving rise to a stable CHO cell line producing IgM-012_GL. IgM-012 and IgM-012_GL could be purified in good yields and were found to be highly soluble (> 10 mg/mL). SDS-PAGE combined with densitometric analysis revealed that purified IgM-012 consists of 68 ± 5% pentamers, 26 ± 3% hexamers and 5 ± 3% dimers (n = 3). Despite the replacement of 17 residues, assembly of IgM-012_GL into pentamers (58 ± 6%) and hexamers (23 ± 1%) is remarkably efficient, with only a moderate increase in dimers amounting to 19 ± 5% (n = 4; Fig. 2). However, it cannot be completely ruled out that the observed small differences in oligomer distribution might exert subtle effects on the biophysical and functional properties of the proteins.
Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.