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Improved chemical synthesis of hydrophobic Aβ peptides using addition of C-terminal lysines later removed by carboxypeptidase B

Authors

  • Saketh Chemuru,

    1. Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • Ravindra Kodali,

    1. Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • Ronald Wetzel

    Corresponding author
    1. Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

ABSTRACT

Many amyloidogenic peptides are highly hydrophobic, introducing significant challenges to obtaining high quality peptides by chemical synthesis. For example, while good yield and purity can be obtained in the solid-phase synthesis of the Alzheimer's plaque peptide Aβ40, addition of a C-terminal Ile-Ala sequence to generate the more toxic Aβ42 molecule creates a much more difficult synthesis resulting in low yields and purities. We describe here a new method that significantly improves the Fmoc solid-phase synthesis of Aβ peptides. In our method, Lys residues are linked to the desired peptide's C-terminus through standard peptide bonds during the synthesis. These Lys residues are then removed post-purification using immobilized carboxypeptidase B (CPB). With this method we obtained both Aβ42 and Aβ46 of superior quality that, for Aβ42, rivals that obtained by recombinant expression. Intriguingly, the method appears to provide independent beneficial effects on both the total synthetic yield and on purification yield and final purity. Reversible Lys addition with CPB removal should be a generally useful method for making hydrophobic peptides that is applicable to any sequence not ending in Arg or Lys. As expected from the additional hydrophobicity of Aβ46, which is extended from the sequence Aβ42 by a C-terminal Thr-Val-Ile-Val sequence, this peptide makes typical amyloid at rates significantly faster than for Aβ42 or Aβ40. The enhanced amyloidogenicity of Aβ46 suggests that, even though it is present in relatively low amounts in the human brain, it could play a significant role in helping to initiate Aβ amyloid formation. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 206–221, 2014.

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