title: An improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies.
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.
authors.affiliation: Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Chhetri
authors.affiliation: Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India.
authors.forename: Tripti
authors.initials: T
authors.lastname: Pandey
authors.affiliation: Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Chinta
authors.affiliation: Department of Biotechnology, National Institute of Technology, Raipur 492010, India. Electronic address: drawanishkr@gmail.com.
authors.forename: Awanish
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India. Electronic address: timir.tripathi@gmail.com.
authors.forename: Timir
authors.initials: T
authors.lastname: Tripathi
mesh_terms: D016229:Amyloid beta-Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D004765:Enteropeptidase; D004926:Escherichia coli / Q000235:genetics; D000431:Ethanol; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
chemical_list: D016229:Amyloid beta-Peptides; D011993:Recombinant Fusion Proteins; D000431:Ethanol; D004765:Enteropeptidase
keywords: Affinity chromatography; Aggregation; Assembly PCR; Aβ peptide; Enterokinase; Ethanol; High-yield recombinant production