Details

Autor(en) / Beteiligte

• Zerweck, Jonathan
• Venkata, Bharat Somireddy
• Pittman, Jay M.
• Srivastava, Atul K.
• Moore, Patrick C.
• Sachleben, Joseph R.
• Thinakaran, Gopal
• Meredith, Stephen C.

Titel

A versatile method for producing labeled or unlabeled Aβ55, Aβ40, and other β-amyloid family peptides

Ist Teil von

• Protein expression and purification, 2019-10, Vol.162, p.72-82

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2019

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• We present a straightforward, versatile method for expressing and purifying β-amyloid (Aβ40) and transmembrane peptides derived from β-amyloid precursor protein (Aβ55). In principle, these methods should be applicable to other types of strongly aggregating peptides. We start with a DNA plasmid encoding a HexaHis tag with a flexible, hydrophilic linker sequence, followed by a cleavage site, and then Aβ peptides. The HexaHis tag rather than a protein fusion partner (e.g., GST) obviates the need for a folded protein in affinity purification. Second, we present two cleavage methods, using either Factor Xa or BNPS-Skatole. Although the latter procedure requires subsequent reduction of the product, we describe methods for minimizing side reactions. Because the use of BNPS-Skatole obviates the need for a folded protein in the cleavage reaction, it is compatible with harsh conditions (e.g., inclusion of detergents and denaturants) needed to solubilize the fusion proteins; such conditions tend to inactivate Factor Xa. Finally, we also describe purification strategies for Aβ40 and Aβ55 using FPLC and/or reverse phase HPLC. Yields of peptide after these BNPS-Skatole cleavage and peptide reduction, though subquantitative, greatly exceed those obtained using Factor Xa cleavage, as the reaction of BNPS-Skatole is insensitive to the presence of detergents and denaturants, and therefore can be used to produce highly aggregative and low solubility peptides such as Aβ55. Trp is a low abundance amino acid in proteins generally, and for peptides like Aβ55, and other transmembane peptides lacking Trp in relevant positions, this cleavage method remains a useful option. •Versatile expression system for Aβ peptides, such as Aβ40 and Aβ55.•Applicable to other highly aggregative peptides/proteins, including TM proteins.•No folded protein needed for fusion partners or (in some cases) enzymatic cleavage.•Uses enzymatic (Factor Xa) or chemical cleavage at Trp residues (BNPS-Skatole).•Useful isotopic labeling for NMR and other applications.