Expression, purification, and characterization of recombinant human beta-amyloid42 peptide in Escherichia coli.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Evidence indicates that abnormal processing and extracellular deposition of the beta-amyloid42 peptide, the longer form of proteolytic derivative of the transmembrane glycoprotein-amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Since it is convenient and economical to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify beta-amyloid42 using glutathione-S-transferase (GST) fusion system. beta-Amyloid42 gene was inserted into a vector pGEX-4T-1 to construct a GST-fusion protein. The fusion protein GST-beta-amyloid42, expressed in BL21 (DE3) strain, was purified with GSH-affinity chromatography followed by thrombin cleavage. The digested product was further purified with an additional GSH-affinity and a Benzamidine chromatography step. After cleavage and purification, the beta-amyloid42 moiety showed the expected size of 4.5 kDa on Tricine-SDS-PAGE, and was further confirmed by Western blot. Moreover, the fibrillar recombinant beta-amyloid42 exhibited great aggregation activity and showed neurotoxicity on neuron cells in vitro. These results suggest that our method will be useful in obtaining a large quantity of recombinant beta-amyloid42 peptide for further physiological and biochemical studies.
Zhang
Li
L
Jiangsu Institute of Nuclear Medicine, Key Laboratory of Nuclear Medicine, Ministry of Health, Wuxi, Jiangsu 214063, China.
Yu
Huixin
H
Song
Cuicui
C
Lin
Xiufeng
X
Chen
Bo
B
Tan
Chen
C
Cao
Guoxian
G
Wang
Zhengwu
Z
D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C075222:amyloid beta-protein (1-42); D005982:Glutathione Transferase