An efficient Escherichia coli expression system for the production of mature-type alkaline serine protease II (mASP II) has been constructed. Complementary deoxyribonucleic acid-encoding mASP II was inserted into the inducible bacterial expression vector pGE-30. After introduction into E. coli, the plasmid was expressed by isopropyl-1-thio-β-d-galactopyranoside, and the recombinant product was purified using a Ni–nitrilotriacetic acid column. The purified product had the expected NH₂-terminal sequence and showed a scrapie isoform of prion protein-degrading activity using hamster scrapie 263K prions as a substrate.