An Efficient Method for the Expression and Purification of Aβ(M1–42)

Abstract

Advances in amyloid research rely on improved access to the β-amyloid peptide, Aβ. N-Terminal methionine-extended Aβ, Aβ(M1-42), is a readily expressed and widely used form of Aβ with properties comparable to those of the natural Aβ(1-42) peptide. Expression of Aβ(M1-42) is simple to execute and avoids an expensive and often difficult enzymatic cleavage step associated with expression and isolation of Aβ(1-42). This paper reports an efficient method for the expression and purification of Aβ(M1-42) and N-labeled Aβ(M1-42). This method affords the pure peptide at ∼19 mg/L of bacterial culture through simple and inexpensive steps in 3 days. This paper also reports a simple method for the construction of recombinant plasmids and the expression and purification of Aβ(M1-42) peptides containing familial mutations. We anticipate that these methods will enable experiments that would otherwise be hindered by insufficient access to Aβ.

Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures

“…To unravel AβO structure–activity relationships, AβOs that faithfully model endogenous aggregates must be readily accessible. Recombinant Aβ(M1–40) represents a potential source of AβOs in this regard, as the monomer can be produced in high yields [24,25,26], and oligomers exhibit potent synaptotoxicity. Based on our hypothesis that the LMW oligomers common to both synthetic WT Aβ40 and recombinant Aβ(M1–40) self-assembly pathways contribute to observed synaptic dysfunction, we examined the quaternary structures of oligomers generated from monomers derived from the two peptides under in vitro self-assembly conditions.…”

“…The recombinant Aβ expression system developed by Walsh and coworkers represents a high-yielding source of Aβ that can be produced quickly and in high purity [24,25,26]. The peptide sequence is called Aβ(M1–40) due to the presence of an N-terminal methionine introduced at a start codon.…”

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

“…Hence, this speed was used to collect the transformed cells from the 1 L cultures. The cells may also be pelleted by centrifuging the cultures at 2800× g if using a JA-10 rotor [14]. Discard the supernatant liquid LB and resuspend the pelleted cells in 25 mL of 1× PBS and transfer the thick cell suspension to a 50 mL falcon tube using a 10 mL pipet.Centrifuge the cells at 7068× g at 4 °C for 25 min and discard the 1× PBS supernatant. PAUSE STEP: Store the pelleted cells at −80 °C until the next day or when ready for cell lysis.…”

“…Typically a relatively large (43 residues) and hydrophobic peptide like Aβ(M1-42) [3] is not separated with a C18 column. However, heating the column between 60–80 °C results in a single peak during separation of pure Aβ(M1-42) peptide with yields similar to alternative protocols [12,13,14]. The lyophilized peptide is characterized by mass spectrometry to verify the identity of the peptide and to detect any impurities present.…”

“…Recently, Yoo et al published a protocol where the Aβ(M1-42) peptide was purified using reverse-phase high-performance liquid chromatography (HPLC) [14]. We have further expanded and optimized this protocol with the following changes to provide improved versatility to the method: 1) We have expressed the pET- Sac-Abeta(M1-42) plasmid in Rosetta(DE3)pLysS cells (a BL21 derivative designed to enhance the expression of eukaryotic proteins) in addition to the BL21(DE3)pLysS cells; 2) We have performed the purification of the peptide with HPLC using the commonly available C18 columns with optimized solvent system conditions; 3) We have provided characteristic details of the peptide with Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) and verified its characteristic aggregate formations in different conditions by western blotting and Atomic Force Microscopy (AFM) to warrant future biological use.…”