A BIOTECHNOLOGICAL METHOD PROVIDES ACCESS TO AGGREGATION COMPETENT MONOMERIC ALZHEIMERS 1-42 RESIDUE AMYLOID PEPTIDE

Senile plaques, a neuropathological hallmark of Alzheimer's disease, c onsist primarily of insoluble aggregates of beta-amyloid peptide (A be ta). A 42-residue peptide (A beta(1-12)) appears to be the predominant form. In contrast to A beta(1-10), A beta(1-12) is characterized by i ts extreme tendency to aggregate into fibers or precipitate. A tailore d biotechnological method prevents aggregation of A beta(1-12) monomer s during its production. The method is based on a protein tail fused t o the amino terminus of A beta. This tail leads to a high expression i n E. coli, and a histidine affinity tag facilitates purification. Sele ctive cleavage of the fusion tail is performed with cyanogen bromide b y immobilizing the fusion protein on a reversed phase chromatography c olumn. Cleavage then occurs only at the methionine positioned at the d esigned site but not at the methionine contained in the membrane ancho r sequence of A beta. Furthermore, immobilization prevents aggregation of cleaved A beta. Elution from the HPLC column and all succeeding pu rification steps are optimized to preserve A beta(1-12) as a monomer. Solutions of monomeric A beta 1-12 spontaneously aggregate into fibers within hours. This permits the investigation of the transition of mon omers into fibers and the correlation of physico-chemical properties w ith biological activities. Mutations of A beta(1-12) at postion 35 inf luence the aggregation properties. Wild-type A beta(1-12) with methion ine at position 35 has similar properties as A beta with a methionine sulfoxide residue. The fiber formation tendency, however, is reduced w hen position 35 is occupied by a glutamine, serine, leucine, or a glut amic acid residue.