期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 113, 期 -, 页码 63-71出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2015.05.002
关键词
Amyloid-beta; Alzheimer's disease; His-tag; TEV protease; Partial refolding; NMR; Metal ions
类别
资金
- Academia Sinica, Taiwan
- National Science Council, Taiwan [NSC 102-2113-M-001-011, MOST 103-2113-M-001-015]
Amyloid-beta (A beta) is a major constituent in the senile plaques of patients with Alzheimer's disease (AD). A beta has been intensively studied in amyloid research; however, challenges posed by data reproducibility arise from purity of synthetic A beta and high expense for its isotope-labeling. The difficulties motivate development and optimization of recombinant A beta (rA beta) production. Here, we report a new procedure to express and purify high quality rA beta 40 from Escherichia coil. The new A beta construct expressed insoluble A beta fused with an N-terminal histidine-tag connected by a linker harboring TEV protease cut site. After purification and partial refolding, the fusion tag was removed by TEV protease cleavage, immobilized metal affinity chromatography (IMAC), and reversed phase-HPLC purification with a yield of 3.5 mg/L culture with and without N-15 label. The rA beta adopts classic amyloid fibrillization and is capable of binding to its clinical relevant metal ions. (C) 2015 Elsevier Inc. All rights reserved.
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