Abstract：
:Amyloid-beta (Aβ) peptide mediates several neurodegenerative diseases. The 42 amino acid (Aβ1-42) is the predominant form of peptide found in the neuritic plaques and has been demonstrated to be neurotoxic in vivo and in vitro. The availability of large quantities of Aβ peptide will help in several biochemical and biophysical studies that may help in exploring the aggregation mechanism and toxicity of Aβ peptide. We report a convenient and economical method to obtain such a peptide biologically. Synthetic oligonucleotides encoding Aβ1-42 were constructed and amplified through the polymerase cycling assembly (also known as assembly PCR), followed by the amplification PCR. Aβ1-42 gene was cloned into pET41a(+) vector for expression. Interestingly, the addition of 3% (v/v) ethanol to the culture medium resulted in the production of large amounts of soluble Aβ fusion protein. The Aβ fusion protein was subjected to a Ni-NTA affinity chromatography followed by enterokinase digestion, and the Aβ peptide was purified using glutathione Sepharose affinity chromatography. The peptide yield was ∼15mg/L culture, indicating the utility of this method for high-yield production of soluble Aβ peptide. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and immunoblotting with anti-His antibody confirmed the identity of purified Aβ fusion protein and Aβ peptide. In addition, this method provides an advantage over the chemical synthesis and other conventional methods used for large-scale production of recombinant Aβ peptide.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Chhetri G,Pandey T,Chinta R,Kumar A,Tripathi Tdoi
10.1016/j.pep.2015.05.015subject
Has Abstractpub_date
2015-10-01 00:00:00pages
71-6eissn
1046-5928issn
1096-0279pii
S1046-5928(15)00152-7journal_volume
114pub_type
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