Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction

Protein Pept Lett. 2008;15(2):119-30. doi: 10.2174/092986608783489599.

Abstract

BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)(6) and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (His)(6) derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 A resolution.

MeSH terms

  • Alzheimer Disease / metabolism
  • Alzheimer Disease / pathology
  • Amino Acid Sequence
  • Amyloid Precursor Protein Secretases / genetics
  • Amyloid Precursor Protein Secretases / isolation & purification*
  • Amyloid Precursor Protein Secretases / metabolism
  • Animals
  • Aspartic Acid Endopeptidases / genetics
  • Aspartic Acid Endopeptidases / isolation & purification*
  • Aspartic Acid Endopeptidases / metabolism
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Crystallization
  • HIV Protease / metabolism*
  • Humans
  • Molecular Sequence Data
  • Molecular Structure
  • Protein Conformation
  • Protein Precursors / genetics
  • Protein Precursors / isolation & purification*
  • Protein Precursors / metabolism
  • Sequence Analysis, Protein
  • X-Ray Diffraction

Substances

  • Protein Precursors
  • Amyloid Precursor Protein Secretases
  • Aspartic Acid Endopeptidases
  • HIV Protease
  • p16 protease, Human immunodeficiency virus 1
  • BACE1 protein, human