Evidence for a key role of beta-amyloid (Aβ) in Alzheimers disease has led to considerable interest in potential therapeutic strategies targeting enzymes involved in processing the amyloid precursor protein (APP). Beta-site APP Cleaving Enzyme (BACE or β-secretase) is a membrane bound aspartyl protease that has been shown to be directly involved in Aβ production and, therefore, is at the forefront of therapeutic targets in the treatment of Alzheimers disease. BACE-2, an enzyme closely related to BACE, regulates Aβ production in a manner antagonistic to BACE, suggesting that non-selective inhibition of BACE-2 by BACE inhibitors might impair the lowering of Aβ. The design of BACE inhibitors that do not inhibit BACE-2 would be enhanced by structural and kinetic studies, efforts that typically demand considerable amounts of both enzymes. A BACE-2 construct containing 19 residues of the BACE prosegment followed by the BACE-2 catalytic domain sequence, Asp36 … ..Trp447, was produced in E. coli inclusion bodies (IB) at 110-140 mg/L cell culture. Exploration of a variety of refolding conditions resulted in an efficient method for refolding the resulting pro- BACE-2 construct, and this protein undergoes facile autocatalytic cleavage, optimal at pH 4, at the Leu40-↓-Ala41 bond. Refolded BACE-2 was purified by anion exchange, molecular sieve, and affinity chromatographies, yielding 105 mg of homogeneous enzyme (kcat/ Km = 1.2 x 104 x M-1 x sec-1) from 8 liters of E. coli cell culture.