Synthesis and characterization of the Kunitz protease-inhibitor domain of the beta-amyloid precursor protein.
To understand the pathological process by which amyloid is deposited in Alzheimer's disease, it is important to characterize the proteolytic processing events of the beta-amyloid precursor protein (beta-APP) from which the amyloid-forming fragment is excised. A potentially important component in beta-APP processing is the 57-amino acid (aa) Kunitz serine protease inhibitor (KPI) located within the extracellular domain of both the 751- and 770-aa isoforms of beta-APP. We have synthesized DNA encoding the 57-aa KPI domain as a necessary step in identifying the role of the protease inhibitor in beta-APP processing and amyloid formation. A bacterial secretion system directed by the alkaline phosphatase signal peptide of Escherichia coli linked to a synthetic gene encoding KPI was used to produce soluble, extracellular recombinant KPI (reKPI) protein. The reKPI protein was purified to homogeneity from bacterial supernatants and was biochemically and biologically characterized. Complete aa sequence analysis confirmed the fidelity of the reKPI, and fast-atom bombardment mass-spectral analysis was used to document that reKPI was of the predicted Mr. The reKPI is as active on a molar basis as the inhibitor-containing beta-APP when assayed for inhibition of trypsin activity. Together these data suggest that reKPI protein is properly folded and lacking in modified aa. Hence, this reKPI will be an important reagent in gaining a better understanding of the role of the KPI domain in beta-APP function and metabolism, as well as in the proteolytic events involved in beta-amyloid formation.
Schilling
J
J
California Biotechnology Inc., Mountain View 94043.
Wang
Y
Y
Lau
K
K
Smith
L
L
Cordell
B
B
D000544:Alzheimer Disease / Q000235:genetics; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000235:genetics* / Q000494:pharmacology; D016564:Amyloid beta-Protein Precursor; D007611:Aprotinin / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin / Q000378:metabolism
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D015345:Oligonucleotide Probes; D011498:Protein Precursors; D011994:Recombinant Proteins; D007611:Aprotinin; D014357:Trypsin