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

Abstract：

: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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Zerweck J,Venkata BS,Pittman JM,Srivastava AK,Moore PC,Sachleben JR,Thinakaran G,Meredith SC

doi

10.1016/j.pep.2019.04.006

subject

Has Abstract

pub_date

2019-10-01 00:00:00

pages

72-82

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(19)30048-8

journal_volume

162

pub_type

杂志文章

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