Expression and purification of amyloid-beta peptides from Escherichia coli

Abstract

Soluble oligomers and fibrillar deposits of amyloid beta (Abeta) are key agents of Alzheimer's disease pathogenesis. However, the mechanism of amyloid aggregation and its interaction with live cells still remain unclear requiring the preparation of large amounts of pure and different Abeta peptides. Here we describe an Escherichia coli expression system using a fusion protein to obtain either Abeta(1-40) or Abeta(1-42) by essentially the same procedure. The fusion protein uses a His-tagged intestinal fatty acid binding protein (IFABP) followed by a six-glycine linker and a Factor Xa cleavage site before the Abeta. The advantages of this system are that the fusion protein can be expressed in large amounts, that the fusion partner, IFABP, has been well characterized in terms of folding, that Abeta or mutated Abeta peptides can be obtained without any extra residues attached to the N-terminus and that the system can be used to incorporate fluorine-labeled amino acids. The incorporation of fluorine-labeled amino acids using auxotrophic strains is a useful NMR probe of side chain behavior. We obtain final yields of 4 and 3mg/L of culture for Abeta(1-40) and Abeta(1-42), respectively.

Figure 1

Construct of the pQE-80L-IFABP-Aβ expression vector.

Figure 2

Diagrammatic scheme of the purification protocol.

Figure 3

SDS PAGE gels of cell extracts. a) IFABP-A _1–40. Lane 1, cell extract supernatant. Lane 2, pellet of the cell extract of dissolved in 8 M urea. Lane 3, molecular weight markers. b) IFABP-A _1–42. Lane 1, cell extract supernatant. Lane 2, pellet of cell extract dissolved in 8 M urea. Lane 3, molecular weight markers. The fusion protein band is circled in Figure 3a and 3b.

Figure 4

SDS Page gels showing Factor Xa cleavage of the fusion proteins. a) IFABP-A _1–40. Lane 1, Ni-NTA column purified fusion protein. Lane 2, after purification with size exclusion chromatography and prior to cleavage with Factor Xa, Lane 3, after cleavage with Factor Xa. Lane 4, molecular weight markers. b) IFABP-A _1–42. Lane 1, Ni-NTA column purified fusion protein. Lane 2, after purification with size exclusion chromatography and prior to cleavage with Factor Xa, Lane 3, after cleavage with Factor Xa. Lane 4, molecular weight markers.

Figure 5

SDS-PAGE gel of purified A _1–40 and A _1–42. Purified A _1–40 (Lane 1), and A _1–42 (Lane 2). Markers shown in Lane 3.

Figure 6

Mass spectrometry of purified A _1–40 and A _1–42. a) Electrospray mass spectrometry of A _1–40. The theoretical mass is 4329 daltons. The peaks at 4351, 4373 and 4393 daltons correspond to one, two and three sodium atoms bound to A _1–40. b) Mass spectrometry of A _1–42. The theoretical mass is 4514 daltons. The peak at 4536 daltons corresponds to presence of one sodium atom bound to A _1–42