Synthesis and characterization of the Kunitz protease-inhibitor domain of the β-amyloid precursor protein
| Autor: | Yu Wang, Barbara Cordell, Leanne Smith, James W. Schilling, Ken Lau |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Signal peptide
Gene isoform Amyloid Sequence analysis Molecular Sequence Data Restriction Mapping law.invention Amyloid beta-Protein Precursor Aprotinin Alzheimer Disease law Sequence Homology Nucleic Acid mental disorders Escherichia coli Genetics medicine Humans Trypsin Amino Acid Sequence Cloning Molecular Protein Precursors Chromatography High Pressure Liquid Serine protease Amyloid beta-Peptides Base Sequence biology General Medicine Recombinant Proteins Protease inhibitor (biology) Biochemistry Enzyme inhibitor Recombinant DNA biology.protein Oligonucleotide Probes Plasmids medicine.drug |
| Zdroj: | Gene. 98:225-230 |
| ISSN: | 0378-1119 |
| DOI: | 10.1016/0378-1119(91)90177-d |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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