Novel semisynthetic method for generating full length β‐amyloid peptides

Abstract

Bacterial expression of full length β-amyloid (Aβ) is problematic because of toxicity and poor solubility of the expressed protein, and a strong tendency of Met35 to become oxidized in inclusion bodies. We have developed a semi-synthetic method in which Aβ 1-29 is expressed in bacteria as part of a fusion protein with a C-terminal intein and Chitin-Binding Domain (CBD). There is also a single residue, N-terminal Met extension. The protein, Met-Aβ 1-29 -Intein-CBD, is well expressed and highly water-soluble. After binding of the expressed protein to Chitin beads, treatment with sodium 2-mercapto-ethane sulfonate (MESNA) yields Met-Aβ 1-29 -MESNA, with a C-terminal thioester suitable for native chemical ligation. Met-Aβ 1-29 -MESNA is first subjected to CNBr cleavage, which removes the N-terminal Met residue, but leaves the thioester intact. We synthesized NH 2 -A30C-Aβ 30-40 , which has an N-terminal Cys residue and is the partner for native chemical ligation with Met-Aβ 1-29 -MESNA. Native chemical ligation proceeds rapidly and efficiently (> 90% yield) to give A30C-Aβ 1-40 . The final step is selective desulfurization using Raney-Ni, which also proceeds rapidly and efficiently (> 90% yield) to give native sequence Aβ 1-40 . Overall, this system is highly efficient, and can yield ≈ 8-10 mg of pure Aβ 1-40 from one liter of bacterial culture medium. This procedure is adaptable for producing other Aβ peptides. We have also expressed an Aβ construct bearing a point mutation associated with one type of familial Alzheimer's Disease, the Iowa mutation, i.e., Met-D23N-Aβ 1-29 -Intein-CBD. Since expression of the intein-containing fusion protein is robust in minimal media as well as standard enriched media, this procedure also can be readily modified for incorporating 15 N or 13 C labels for NMR. Future work will also include extending this system to longer Aβ peptides, such as Aβ 1-42 .

DMSO affects Aβ1–40's conformation and interactions with aggregation inhibitors as revealed by NMR

“…This point is relevant as oxidation of Met35 is believed to inhibit Ab aggregation. 12 The 1 HN and 1 Ha d values determined here resemble those reported previously for Ab 1-40 in 95% DMSO/5% dichloroacetic acid 13 and Ab 1-28 in neat DMSO 14 (ESI Fig. S4 †).…”

“…Many biophysical and preclinical studies require the amyloid b peptide (Ab) to be monomeric and unfolded at the start of the experiment. However, since Ab has a strong tendency to oligomerize in aqueous solution, several protocols [1][2][3] based on inteins or click chemistry have been developed to ensure that it is unfolded and monomeric. However, these approaches require special materials and expertise.…”

A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides

“…Aβ peptide recombinant production was attempted in yeast [ 11 ] or by combining recombinant and synthetic procedures [ 12 ] but, to date, the most common expression system remains Escherichia coli . Most often, peptides are appended to a fusion protein that enhance their solubility and ease their isolation and purification using affinity chromatography.…”

Chemical Strategies for Controlling Protein Folding and Elucidating the Molecular Mechanisms of Amyloid Formation and Toxicity

“…To overcome these limitations, Bockhorn et al applied a semisynthetic strategy 77 (similar in design to the one described above for α-syn) in which the 1-29 fragment of Aβ was recombinantly expressed as an intein-chitin binding domain fusion protein, which remained fully soluble but retained the initiating methionine residue and was thus referred to as Met-Aβ . Expression was efficient in both normal and minimal media, ensuring the applicability of this method for preparing isotopically labeled Aβ.…”

“…77 It also offers the possibility of preparing Aβ peptides with PTMs in the C-terminal domain. For example, while oxidation of Met35 to sulfoxide, which decreases Aβ toxicity and selfassembly, has been well investigated, 179 the rarely studied sulfone form was recently found to have profoundly opposite effects.…”