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.