title: Recombinant insect cell expression and purification of human beta-secretase (BACE-1) for X-ray crystallography.
abstract: Human beta-secretase (BACE-1) is a type I integral membrane aspartic protease that catalyzes the internal cleavage of the amyloid precursor protein (APP), generating the N-terminus of the Abeta peptide. The generation and subsequent extracellular deposition of Abeta(1-42) peptide into amyloid plaques in the brain constitute one of the hallmarks of Alzheimer's disease (AD), a common debilitating neurodegenerative disorder. Inhibition of BACE-1 is considered an excellent therapeutic strategy against AD. To generate pure enzyme for protein crystallography and subsequent structure-based drug design, we have expressed a soluble, unglycosylated, 6xHis-tagged form of proBACE-1 in insect cells using baculovirus infection. To avoid production of a mixture of the pro-enzyme form and the mature form of BACE-1, the proprotein convertase furin was coexpressed with proBACE-1, leading to almost complete proteolytic activation of the recombinant enzyme. The mature enzyme was secreted in the conditioned medium of BACE-1/furin coinfected HighFive insect cells. Secreted BACE-1 protein was purified to homogeneity from the medium using subsequent Ni-chelate affinity chromatography, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. To avoid autoproteolysis, all purification steps were performed at pH values outside the activity range of BACE-1. The purified, biologically active enzyme was homogeneous on SDS/PAGE and had the expected sequence and molecular mass determined by N-terminal amino acid sequencing and mass spectrometry, respectively. Moreover, the preparation showed a single peak of the expected size with only 17% polydispersity using dynamic light scattering analysis. The yield of BACE-1 from fermentation cultures was approximately 0.1mg pure enzyme per liter of cell culture medium. The purified protein was successfully used to generate BACE-1/inhibitor co-crystals and to determine the crystal structure of the complex by X-ray analysis. The availability of substantial quantities of active, homogeneous enzyme will be of great help in future structure-based drug design efforts in the search for efficient protease inhibitor drugs to treat AD.
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D005285:Fermentation; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera / Q000166:cytology* / Q000821:virology
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human