Improved chemical synthesis of hydrophobic Aβ peptides using addition of C-terminal lysines later removed by carboxypeptidase B.
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.
Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA.
Saketh
S
Chemuru
Ravindra
R
Kodali
Ronald
R
Wetzel
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D043424:Carboxypeptidase B / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004800:Enzymes, Immobilized / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D066329:Protein Aggregates; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D016229:Amyloid beta-Peptides; D004800:Enzymes, Immobilized; D066329:Protein Aggregates; D043424:Carboxypeptidase B; D008239:Lysine
amyloid; amyloid-β; carboxypeptidase B; high performance liquid chromatography; hydrophobic; immobilized enzyme; lysine; purification; purity; recombinant; solid-phase peptide synthesis; yield; β-sheet