科研之友
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摘要
Amyloid-beta (A beta) peptide mediates several neurodegenerative diseases. The 42 amino acid (A beta(1-42)) 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 A beta peptide will help in several biochemical and biophysical studies that may help in exploring the aggregation mechanism and toxicity of A beta peptide. We report a convenient and economical method to obtain such a peptide biologically. Synthetic oligonucleotides encoding A beta(1-42) were constructed and amplified through the polymerase cycling assembly (also known as assembly PCR), followed by the amplification PCR. A beta(1-42) 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 A beta fusion protein. The A beta fusion protein was subjected to a Ni-NTA affinity chromatography followed by enterokinase digestion, and the A beta peptide was purified using glutathione Sepharose affinity chromatography. The peptide yield was similar to 15 mg/L culture, indicating the utility of this method for high-yield production of soluble A beta peptide. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and immunoblotting with anti-His antibody confirmed the identity of purified A beta fusion protein and A beta peptide. In addition, this method provides an advantage over the chemical synthesis and other conventional methods used for large-scale production of recombinant A beta peptide.
- 出版日期2015-10
