Expression, purification and characterization of a synthetic gene encoding human amyloid beta (Abeta1-42) in Escherichia coli.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Existing evidence indicates that abnormal processing and extracellular deposition of the longer form of the amyloid peptide Abeta(1-42), a proteolytic derivative of the amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Active immunization with Abeta(1-42) has been shown to decrease brain beta deposition and improve cognitive performance in mouse models of AD. In the present study, we sought to express the synthetic gene encoding AB in Escherichia coli to enable rapid production of the antigen and its purification. The synthetic gene has been constructed from six oligonucleotides by employing overlapping PCR strategy and expressed in E. coli using the T7 promoter system. The recombinant peptide has been purified to homogeneity by a single step Ni+2 affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-Abeta(1-42) sera confirms that the corresponding linear B-cell epitopic sequences are available for immunorecognition in the recombinant peptide. This methodology enables rapid, continuous production and purification in bulk amounts of human Abeta sequence by employing bacterial expression system
Subramanian
Sarada
S
Department of Neurochemistry, National Institute of Mental Health & Neurosciences, Bangalore 560 029, India. sarada@nimhans.kar.nic.in
Shree
A N Divya
AN
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D005813:Genes, Synthetic; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C075222:amyloid beta-protein (1-42)