AI Article Synopsis
- The expression and purification of human beta-secretase (BACE1) in bacteria have faced challenges like solubility issues, uneven N-terminus, and inactive enzymes.
- Various mature forms of BACE1 have been successfully expressed in *E. coli*, leading to the development of a protocol to solubilize, refold, and purify these proteins, resulting in homogeneous solutions.
- Activity assays of these BACE1 variants demonstrate enzymatic activity with a kcat of 9.3 min(-1) and a KM of 55 microM using a specific peptide substrate designed to mimic the beta-secretase cleavage sequence.
Article Abstract
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.
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| http://dx.doi.org/10.1016/j.pep.2003.12.014 | DOI Listing |
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