Recombinant expression of amyloid beta 1-42 (AÎ²1-42) in Escherichia coli bacterial system

Badiri, Pouran; Sadeghizadeh, Majid; Bambai, Bijan; Bathaie, S. Zahra; Bahrainy, Mehrnaz; Minuchehr, Zarrin

Recombinant expression of amyloid beta 1-42 (AÎ²1-42) in Escherichia coli bacterial system

Document Type : Original Research

Authors

Pouran Badiri ¹

Majid Sadeghizadeh ¹

Bijan Bambai ²

S. Zahra Bathaie ³

Mehrnaz Bahrainy ²

Zarrin Minuchehr ²

¹ Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University

² Department of Systems Biotechnology, National Institute of Genetic Engineering and Biotechnology

³ Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University

Abstract

Introduction: Amyloid beta (Aβ) is the major constituent of harmful plaques in the Alzheimer’s patients. Thus, study of Aβ and understanding its related molecular and cellular mechanisms is essential for diagnosis and therapeutic interventions. This study introduces a rapid, simple, and cost-effective technique for production and purification of this peptide, utilizing the expression of Aβ gene within bacterial system.

Materials and methods: Aβ gene was synthesized and transferred into the expression vector pET26b. After induction by Lactose and 24 hours of incubation for Aβ expression the cell sediment was analyzed for presence of recombinant peptide using SDS-PAGE and Western blot. Then the purification of recombinant peptide was carried using nickel chloride affinity chromatography. Characterization of purified Aβ was performed by evaluating cell cytotoxicity in 25 µM and 50 µM concentrations using MTT assay on Alzheimer cell line model SH-SY5Y.

Results: Colony PCR and sequencing results showed the correct insertion of Aβ coding fragment into the expression vector. Presence of bands with the expected size in the results of SDS PAGE and western blot had confirmed successful expression of his-tagged recombinant peptide. MTT assay results showed the purified peptide has respectively 30 and 50% cytotoxicity for 25 µM and 50 µM concentrations.

Discussion: Production of amyloid beta peptide in bacterial hosts seems to be favorable. Obtaining Aβ peptide in soluble phase is an important advantage of this study. Hence according to toxicity of the purified peptide, it can be utilized for cell line treatments and further researches on Alzheimer disease.

Keywords

amyloid beta

Peptide

recombinant

Ni-NTA

western blot

Subjects

Industrial Biotechnology

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