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

Abstract

An improved production and purification method for Alzheimer’s disease related methionine-modified amyloid-β 1–40 and 1–42 peptides is proposed, taking advantage of the formation of inclusion body in Escherichia coli. A Thioflavin-S assay was set-up to evaluate inclusion body formation during growth and optimize culture conditions for amyloid-β peptides production. A simple and fast purification protocol including first the isolation of the inclusion bodies and second, two cycles of high pH denaturation/ neutralization combined with an ultrafiltration step on 30-kDa cut-off membrane was established. Special attention was paid to purity monitoring based on a rational combination of UV spectrophotometry and SDS-PAGE analyses at the various stages of the process. It revealed that this chromatography-free protocol affords good yield of high quality peptides in term of purity. The resulting peptides were fully characterized and are appropriate models for highly reproducible in vitro aggregation studies.

Amyloidogenicity and Cytotoxicity of a Recombinant C-Terminal His6-Tagged Aβ1–42

“…3D images of the two artifacts are also shown in Figure S5, which present a visual view of the fibrils. The AFM results were consistent with the ThT fluorescence data and our previous studies, both of which indicated that the recombinant Aβ 1–42 -His 6 shows biochemical and biophysical behaviors consistent with the aggregation characteristics of previously reported recombinant and synthetic Aβ 1–42 preparations. ,, …”

“…The AFM results were consistent with the ThT fluorescence data and our previous studies, 42 both of which indicated that the recombinant Aβ 1−42 -His 6 shows biochemical and biophysical behaviors consistent with the aggregation characteristics of previously reported recombinant and synthetic Aβ 1−42 preparations. 13,43,44 Circular Dichroism Spectroscopy. The secondary structures of Aβ 1−42 -His 6 were examined by far-UV CD spectroscopy after incubation at 37 °C for 24 h. Figure 4a shows the CD spectra of Aβ 1−42 -His 6 and the synthetic peptide after incubation, presented a major maximum at ∼202 nm and a minor minimum around 220 nm, which are characteristic of the β-sheet-rich fibrillar structure.…”

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid beta 1-42

“…Both these methods require additional steps from which Aβ42 has to be eventually cleaved requiring extra time, reagents, as well as comprising on the final peptide yield. Another method used NaOH treatment followed by ultracentrifugation to isolate and purify the insoluble inclusion bodies expressed in the E. coli [20]. This purification method reduces the peptide purity and yield (~ 4 mg of peptide obtained).…”

Identification of a HTT-specific binding motif in DNAJB1 essential for suppression and disaggregation of HTT

“…Aβ42. Aβ42 peptide was overexpressed and purified as described previously 49 . In all, 50 ng of pET-M-Aβ42 plasmid were transformed into E. coli BL21-CodonPlus (DE3)-RIPL cells (Agilent, #230280).…”