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Improving the anti-toxin abilities of the CMG2-Fc fusion protein with the aid of computational design.

Xi Y, Wu X, Gao L, Shao Y, Peng H, Chen H, Chen H, Hu X, Yue J - PLoS ONE (2014)

Bottom Line: An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections.This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable.Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing, China.

ABSTRACT
CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

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Related in: MedlinePlus

The purified CMG2-Fc mutants were analyzed by 10% SDS-PAGE under reducing conditions (A, B) or non-reducing conditions (C).Gels were visualized by Coomassie blue staining (A, C) or subjected to western blotting with a mouse anti-CMG2 antibody (B). Lanes 1, 4 and 6: CMG2-Fc (Y158Q); lanes 2, 5 and 7: CMG2-Fc (E117Q); lanes 3 and 8: protein marker.
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pone-0104674-g003: The purified CMG2-Fc mutants were analyzed by 10% SDS-PAGE under reducing conditions (A, B) or non-reducing conditions (C).Gels were visualized by Coomassie blue staining (A, C) or subjected to western blotting with a mouse anti-CMG2 antibody (B). Lanes 1, 4 and 6: CMG2-Fc (Y158Q); lanes 2, 5 and 7: CMG2-Fc (E117Q); lanes 3 and 8: protein marker.

Mentions: CMG2-Fc is a glycoprotein and must be expressed in eukaryotic cells, such as the CHO cell line. CHO cells are the most dependable eukaryotic host cells for the industrial production of therapeutic proteins due to their appropriate product secretion post-translational processing, a reasonable safety profile and the approval of regulatory authorities [28]. The CHO-S cell line is a stable cell line sub-cloned from the common CHO cells. The most pertinent advantage of this cell line is that it can be adapted to suspension culture and therefore reduce costs. A recombinant CHO-S cell line producing CMG2-Fc mutant was selected for culture in a 500 ml shake flask (Corning) with serum-free DMEM/F12 medium. The proteins were purified by protein A and identified by SDS-PAGE and western blotting (Figure. 3). The CMG2-Fc mutant is comprised of amino acids 1–225 of human CMG2, followed by the hinge and Fc regions of human IgG1 (the C-terminal 232 amino acids of IgG1). The molecular weight of this fusion was approximately 47 kDa under reducing conditions (consistent with CMG2-Fc) as determined by mass spectroscopy (data not shown). The expression of protein was identified by Western blot analysis using mouse anti-CMG2 polyclonal antibody (Anti-ANTXR2 antibody ab70499, Abcam, UK).


Improving the anti-toxin abilities of the CMG2-Fc fusion protein with the aid of computational design.

Xi Y, Wu X, Gao L, Shao Y, Peng H, Chen H, Chen H, Hu X, Yue J - PLoS ONE (2014)

The purified CMG2-Fc mutants were analyzed by 10% SDS-PAGE under reducing conditions (A, B) or non-reducing conditions (C).Gels were visualized by Coomassie blue staining (A, C) or subjected to western blotting with a mouse anti-CMG2 antibody (B). Lanes 1, 4 and 6: CMG2-Fc (Y158Q); lanes 2, 5 and 7: CMG2-Fc (E117Q); lanes 3 and 8: protein marker.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125234&req=5

pone-0104674-g003: The purified CMG2-Fc mutants were analyzed by 10% SDS-PAGE under reducing conditions (A, B) or non-reducing conditions (C).Gels were visualized by Coomassie blue staining (A, C) or subjected to western blotting with a mouse anti-CMG2 antibody (B). Lanes 1, 4 and 6: CMG2-Fc (Y158Q); lanes 2, 5 and 7: CMG2-Fc (E117Q); lanes 3 and 8: protein marker.
Mentions: CMG2-Fc is a glycoprotein and must be expressed in eukaryotic cells, such as the CHO cell line. CHO cells are the most dependable eukaryotic host cells for the industrial production of therapeutic proteins due to their appropriate product secretion post-translational processing, a reasonable safety profile and the approval of regulatory authorities [28]. The CHO-S cell line is a stable cell line sub-cloned from the common CHO cells. The most pertinent advantage of this cell line is that it can be adapted to suspension culture and therefore reduce costs. A recombinant CHO-S cell line producing CMG2-Fc mutant was selected for culture in a 500 ml shake flask (Corning) with serum-free DMEM/F12 medium. The proteins were purified by protein A and identified by SDS-PAGE and western blotting (Figure. 3). The CMG2-Fc mutant is comprised of amino acids 1–225 of human CMG2, followed by the hinge and Fc regions of human IgG1 (the C-terminal 232 amino acids of IgG1). The molecular weight of this fusion was approximately 47 kDa under reducing conditions (consistent with CMG2-Fc) as determined by mass spectroscopy (data not shown). The expression of protein was identified by Western blot analysis using mouse anti-CMG2 polyclonal antibody (Anti-ANTXR2 antibody ab70499, Abcam, UK).

Bottom Line: An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections.This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable.Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing, China.

ABSTRACT
CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

Show MeSH
Related in: MedlinePlus