High Yield Expression of Human BACE Constructs in Eschericia coli for Refolding, Purification, and High Resolution Diffracting Crystal Forms
| Autor: | A. Tomasselli, D. Paddock, T. Emmons, A. Mildner, J. Leone, J. Lull, J. Cialdella, D. Prince, H. Fischer, R. Heinrikson, T. Benson, A. G. Tomasselli, D. J. Paddock, T. L. Emmons, A. M. Mildner, J. W. Leone, J. M. Lull, J. I. Cialdella, D. B. Prince, H. D. Fischer, R. L. Heinrikson, T. E. Benson |
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| Rok vydání: | 2008 |
| Předmět: |
Protein Folding
Protein Conformation Molecular Sequence Data Peptide CHO Cells Biochemistry Inclusion bodies Cricetulus Protein structure HIV Protease X-Ray Diffraction Affinity chromatography Alzheimer Disease Structural Biology Cricetinae Escherichia coli Animals Aspartic Acid Endopeptidases Humans Amino Acid Sequence Protein Precursors Peptide sequence chemistry.chemical_classification Molecular Structure General Medicine Recombinant Proteins Transmembrane domain Crystallography Enzyme Ectodomain chemistry Amyloid Precursor Protein Secretases Crystallization |
| Zdroj: | Protein & Peptide Letters. 15:131-143 |
| ISSN: | 0929-8665 |
| Popis: | BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments. |
| Databáze: | OpenAIRE |
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