Production of recombinant amyloid-beta peptide 42 as an ubiquitin extension.
Amyloid-beta peptide 42 (Abeta42) mediates neuronal degeneration in Alzheimer's disease (AD). We sought to produce recombinant Abeta42 as an ubiquitin extension. A synthetic oligonucleotide encoding Abeta42 was constructed and cloned as an extended polypeptide of hexahistidine-tagged ubiquitin (H(6)Ub) using the pET vector. Isopropyl-beta-D-thiogalactopyranoside induction of transformed Escherichia coli resulted in the production of large amounts of insoluble H(6)Ub-Abeta42 fusion protein. H(6)Ub-Abeta42 was solubilized in 8 M urea and applied to a nickel-nitrilotriacetic acid affinity column for purification. Column washing removed the urea and soluble H(6)Ub-Abeta42 was eluted, indicating that covalently attached ubiquitin prevented Abeta42 from aggregating. Abeta42 was cleaved from H(6)Ub using recombinant yeast ubiquitin hydrolase-1 (YUH-1) and purified using reverse-phase chromatography. The recombinant Abeta42 prepared in this study has the same toxic effect on human neuroblastoma SH-SY5Y cells comparing with chemically synthesized, commercial one. The peptide yield was more than 4 mg/L culture, indicating this ubiquitin fusion technique is an attractive method for production of aggregation-prone peptides such as Abeta42.
Lee
Eun Kyung
EK
Department of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea.
Hwang
Jin Ha
JH
Shin
Dong Yeon
DY
Kim
Dae Ihn
DI
Yoo
Yung Joon
YJ
D016229:Amyloid beta-Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D045744:Cell Line, Tumor; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D025801:Ubiquitin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C075222:amyloid beta-protein (1-42)