A biotechnological method provides access to aggregation competent monomeric Alzheimer's 1-42 residue amyloid peptide.
Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of beta-amyloid peptide (A beta). A 42-residue peptide (A beta 1-42) appears to be the predominant form. In contrast to A beta 1-40, A beta 1-42 is characterized by its extreme tendency to aggregate into fibers or precipitate. A tailored biotechnological method prevents aggregation of A beta 1-42 monomers during its production. The method is based on a protein tail fused to the amino terminus of A beta. This tail leads to a high expression in E. coli, and a histidine affinity tag facilitates purification. Selective cleavage of the fusion tail is performed with cyanogen bromide by immobilizing the fusion protein on a reversed phase chromatography column. Cleavage then occurs only at the methionine positioned at the designed site but not at the methionine contained in the membrane anchor sequence of A beta. Furthermore, immobilization prevents aggregation of cleaved A beta. Elution from the HPLC column and all succeeding purification steps are optimized to preserve A beta 1-42 as a monomer. Solutions of monomeric A beta 1-42 spontaneously aggregate into fibers within hours. This permits the investigation of the transition of monomers into fibers and the correlation of physico-chemical properties with biological activities. Mutations of A beta 1-42 at position 35 influence the aggregation properties. Wild-type A beta 1-42 with methionine at position 35 has similar properties as A beta with a methionine sulfoxide residue. The fiber formation tendency, however, is reduced when position 35 is occupied by a glutamine, serine, leucine, or a glutamic acid residue.
Döbeli
H
H
Pharma Division, F. Hoffmann-La Roche, Basel, Switzerland. heinz.doebeli@roche.com
Draeger
N
N
Huber
G
G
Jakob
P
P
Schmidt
D
D
Seilheimer
B
B
Stüber
D
D
Wipf
B
B
Zulauf
M
M
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010963:Plasmodium falciparum / Q000737:chemistry; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012996:Solutions
D016229:Amyloid beta-Peptides; D000953:Antigens, Protozoan; D011993:Recombinant Fusion Proteins; D012996:Solutions; D003488:Cyanogen Bromide