A general procedure for the purification of human β-secretase expressed in Escherichia coli
| Autor: | Paul L. Darke, Sanjeev Munshi, Zhongguo Chen, Lawrence C. Kuo, Bei Xu, Joan Zugay-Murphy, V V Sardana, Mohinder K. Sardana |
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| Rok vydání: | 2004 |
| Předmět: |
Protein Folding
Molecular Sequence Data Receptors Cell Surface medicine.disease_cause Inclusion bodies Substrate Specificity Amyloid beta-Protein Precursor Endopeptidases Escherichia coli Amyloid precursor protein medicine Aspartic Acid Endopeptidases Humans Amino Acid Sequence Enzyme kinetics Peptide sequence biology Enzyme Activation Protease Nexins Transmembrane domain Biochemistry Mutagenesis Site-Directed biology.protein Protein folding Amyloid Precursor Protein Secretases Carrier Proteins Crystallization Amyloid precursor protein secretase Biotechnology |
| Zdroj: | Protein Expression and Purification. 34:190-196 |
| ISSN: | 1046-5928 |
| Popis: | 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 N(670)L(671) substituting for the residues K(670)M(671), reveal a kcat and KM of 9.3 min(-1) and 55 microM, respectively. |
| Databáze: | OpenAIRE |
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