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 portion (CT105) including beta/A4 domain were highly expressed using strong expression systems in E. coli. These recombinant APP peptides were purified with a combination of urea solubilization and ion-exchange chromatography and used for proteolytic processing by thrombin. Three thrombin cleavage sites were predicted by the decrease of APP770 and the appearance of M(r) 56, 27 and 18 kDa fragments containing beta/A4 domain on SDS-PAGE gel and on the immunoblot. A similar but limited proteolysis of platelet APPs exposed to thrombin resulted in the stimulated production of 60 and 27 KDa carboxyl terminal peptides containing the intact beta/A4. This thrombin mediated proteolysis was completely blocked by hirudin, the specific thrombin inhibitor. These results suggest that thrombin may play a role in altered processing of APP to generate potentially amyloidogenic intermediates in vivo leading to amyloid deposition.
Chong
Y H
YH
Department of Pharmacology, College of Medicine, Seoul National University, Korea.
Jung
J M
JM
Choi
W
W
Park
C W
CW
Choi
K S
KS
Suh
Y H
YH
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014508:Urea
D016564:Amyloid beta-Protein Precursor; D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014508:Urea; D004247:DNA; D013917:Thrombin