BACTERIAL EXPRESSION, PURIFICATION OF FULL-LENGTH AND CARBOXYL-TERMINAL FRAGMENT OF ALZHEIMER AMYLOID PRECURSOR PROTEIN AND THEIR PROTEOLYTIC PROCESSING BY THROMBIN

Human amyloid protein precursor(APP770) and its carboxyl terminal port ion (CT105) including beta/A4 domain were highly expressed using stron g expression systems in E. coli. These recombinant APP peptides were p urified with a combination of urea solubilization and ion-exchange chr omatography and used for proteolytic processing by thrombin. Three thr ombin cleavage sites were predicted by the decrease of APP770 and the appearance of Mr 56, 27 and 18 KDa fragments containing beta/A4 domain on SDS-PAGE get and on the immunoblot. A similar but limited proteoly sis of platelet APPs exposed to thrombin resulted in the stimulated pr oduction of 60 and 27 KDa carboxyl terminal peptides containing the in tact beta/A4. This thrombin mediated proteolysis was completely blocke d by hirudin, the specific thrombin inhibitor. These results suggest t hat thrombin may play a role in altered processing of APP to generate potentially amyloidogenic intermediates in vivo leading to amyloid dep osition.