HIGH-LEVEL EXPRESSION AND IN-VITRO MUTAGENESIS OF A FIBRILLOGENIC 109-AMINO-ACID C-TERMINAL FRAGMENT OF ALZHEIMERS-DISEASE AMYLOID PRECURSOR PROTEIN

We amplified DNA encoding the 3' 109 codons of Alzheimer's-disease amy loid precursor protein (APP) inclusive of the beta protein (Abeta) and cytoplasmic domains from cDNA using oligonucleotide primers designed to facilitate cloning into the T7 expression vector pT7Ad23K13. We als o modified this construct to generate recombinant molecules incorporat ing two recently described APP mutants by site-directed mutagenesis. B oth native C109 (deletion construct inclusive of the C-terminal 109 re sidues of APP) and constructs with a single mutation at codon 642 (T - -> G, resulting in a substitution of glycine for valine) or a double m utation at codons 595 (G --> T, substituting asparagine for lysine) an d 596 (A --> C, substituting leucine for methionine) were expressed in Escherichia coli to levels of 5-20 % of total bacterial protein after induction. The major constituent of expressed C109 protein had an app arent molecular mass of 16-18 kDa by SDS/PAGE and appeared to be the f ull-length construct by size and N-terminal microsequencing. Also pres ent was a 4-5 kDa species that co-purified with C109, constituting onl y approximately 1 % of expressed protein, which was revealed by Wester n-blot analysis with antibodies specific for Abeta epitopes and after biotinylation of purified recombinant C109. This fragment shared N-ter minal sequence with, and appeared to arise by proteolysis of, full-len gth C109 in biosynthetic labelling experiments. C109 spontaneously pre cipitated after dialysis against NaCl or water, and with prolonged (> 20 weeks) standing was found by electron microscopy to contain a minor (< 5 %) fibrillar component that was reactive with antibodies to a C- terminal epitope of APP. Recombinant C109 appears to duplicate some of the biochemical and physicochemical properties of C-terminal Abeta-in clusive fragments of APP that have been found in transfected cells, br ain cortex and cerebral microvessels.