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Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Chromikova V, Mader A, Hofbauer S, Göbl C, Madl T, Gach JS, Bauernfried S, Furtmüller PG, Forthal DN, Mach L, Obinger C, Kunert R - Biochim. Biophys. Acta (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.

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Related in: MedlinePlus

SDS-PAGE of 2G12-IgM. Purified IgM-012, IgM-012_GL and IgM-617 (control) were separated on 3–12% gradient NuPage® Bis-Tris gels and detected by silver staining.
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f0010: SDS-PAGE of 2G12-IgM. Purified IgM-012, IgM-012_GL and IgM-617 (control) were separated on 3–12% gradient NuPage® Bis-Tris gels and detected by silver staining.

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.


Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Chromikova V, Mader A, Hofbauer S, Göbl C, Madl T, Gach JS, Bauernfried S, Furtmüller PG, Forthal DN, Mach L, Obinger C, Kunert R - Biochim. Biophys. Acta (2015)

SDS-PAGE of 2G12-IgM. Purified IgM-012, IgM-012_GL and IgM-617 (control) were separated on 3–12% gradient NuPage® Bis-Tris gels and detected by silver staining.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582045&req=5

f0010: SDS-PAGE of 2G12-IgM. Purified IgM-012, IgM-012_GL and IgM-617 (control) were separated on 3–12% gradient NuPage® Bis-Tris gels and detected by silver staining.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.

Show MeSH
Related in: MedlinePlus