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Biopolymers ; 102(2): 206-21, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24488729

RESUMO

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 Aß40, addition of a C-terminal Ile-Ala sequence to generate the more toxic Aß42 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 Aß 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 Aß42 and Aß46 of superior quality that, for Aß42, 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 Aß46, which is extended from the sequence Aß42 by a C-terminal Thr-Val-Ile-Val sequence, this peptide makes typical amyloid at rates significantly faster than for Aß42 or Aß40. The enhanced amyloidogenicity of Aß46 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 Aß amyloid formation.


Assuntos
Peptídeos beta-Amiloides/síntese química , Carboxipeptidase B/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lisina/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/isolamento & purificação , Peptídeos beta-Amiloides/ultraestrutura , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Enzimas Imobilizadas/metabolismo , Cinética , Dados de Sequência Molecular , Agregados Proteicos , Espectrometria de Massas por Ionização por Electrospray , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura
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