Expression and purification of amyloid-beta peptides from Escherichia coli.
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.
Garai
Kanchan
K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Crick
Scott L
SL
Mustafi
Sourajit M
SM
Frieden
Carl
C
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D050556:Fatty Acid-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D050556:Fatty Acid-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)