An Efficient Method for Expression and Purification of Active Human IGF-1 in Escherichia Coli

Publish Year: 1398
نوع سند: مقاله کنفرانسی
زبان: English
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RROYAN20_127

تاریخ نمایه سازی: 29 مهر 1398

Abstract:

Background: The generation of neurons in the adult mamma-lian brain requires the activation of quiescent neural stem cells (NSCs). This activation and the sequential steps of neuron for-mation from NSCs are regulated by a number of stimuli such as growth factors. Insulin-like growth factor-1 (IGF-1) is one of these factors that exerts pleiotropic effects and regulates multi-ple cellular processes based on its concentration, cell type and the developmental stage of target cells. This factor shows troph-ic effects on neuronal regeneration in the central and periph-eral nervous systems. It stimulates protein synthesis in neurons, glia, oligodendrocytes and Schwann cells that result in inhibi-tion of cell apoptosis. Human IGF-1 is a non-glycosylated poly-peptide hormone containing 70 amino acids in a single chain with three intramolecular disulfide bonds. Recently, several variants of engineered human IGF-1 have been produced in or-der to improve the protein stability, activity and tighter binding to IGF-1 receptor. However, the major challenges in production of recombinant human IGF-1 are the yield, purification and the activity of produced peptide as well as the cost of production. In this study, an efficient method was employed to produce active human IGF-1, which was purified without any solubilizing tag. Materials and Methods: The gene of tobacco etch virus (TEV) protease and coding sequence of hIGF-1 with a His-tag, which was fused to TEVsite and Trx (Thioredoxin) gene were cloned into two cloning sites of pET-Duet vector under control of tac and T7 promoters respectively. The vector was transformed into SHuffle T7 competent E. coli and expression condition was optimized for expressing active TEV and Trx/TEVsite/His-tag/hIGF-1 fused protein simultaneously. Afterwards, one-step purification procedure was employed to purify His-tag/hIGF-1 using Ni-NTA resin. Western blot was used to confirm the accu-racy of the purified protein and its functionality was examined on NIH3T3 cells.Results: Our results demonstrated that TEV protease digested its specific site between Trx and hIGF-1 in bacterial cells and the soluble His-tag/hIGF-1 was extracted without Trx-tag. Fol-lowed by, the effect of purified protein on NIH3T3 cells prolif-eration showed that the recombinant protein was functional and folded correctly.Conclusion: This research developed an efficient method to produce soluble and active recombinant hIGF-1 in E. coli, which was purified without any solubilizing tag using a simple, feasible procedure.

Keywords:

Neural Stem Cells (Nscs) , Insulin-Like Growth Fac-tor-1 (IGF-1) , Tobacco Etch Virus (TEV) Protease , Purification , Recombinant

Authors

S Siavashi

Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran. Department of Biology, ACECR Institute of Higher Education, ACECR Institute of Higher Education, Isfahan, Iran

F Aboutalebi

Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

MH Nasr Esfahani

Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

R Khayam Abed

Department of Biology, ACECR Institute of Higher Education, ACECR Institute of Higher Education, Isfahan, Iran. Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran