Expression and purification of human beta-secretase (BACE1) in bacteria have been plagued with issues concerning solubility, inhomogeneous N-terminus, and lack of enzymic activity. Several forms of the mature human BACE1 have been expressed in Escherichia coli with different N-terminal extensions and without the C-terminus transmembrane domain. Although each of the proteins expresses in inclusion bodies, a generalized protocol has been developed to solubilize, refold, and purify these BACE1 variants. The resultant proteins are homogeneous and monodispersed in solution. Each possesses a unique N-terminus. Activity assays using the peptide substrate 7-methoxycoumarin-4-yl-SEVNLDAEFK-2,4-dinitrophenyl-RR, corresponding to the beta-secretase cleavage sequence in the amyloid precursor protein with the Swedish mutations of (NN671)-N-670 substituting for the residues (KM671)-M-670, reveal a k(cat) and K-M of 9.3 min(-1) and 55 muM, respectively. (C) 2004 Elsevier Inc. All rights reserved.