Expression, Purification, Characterization and &lt;i&gt;In Vitro&lt;/i&gt; Activity of Recombinant Mouse Cu/Zn-Binding Superoxide Dismutase (mSOD1)

A Li,F Wang,H Sun,L Huang,Q Wu,R Jiang,Y Liu,Z Luo,Z Zhang

doi:10.4314/tjpr.v12i3.9

Expression, Purification, Characterization and <i>In Vitro</i> Activity of Recombinant Mouse Cu/Zn-Binding Superoxide Dismutase (mSOD1)

A Li, F Wang + Show 7 more

Open Access

https://doi.org/10.4314/tjpr.v12i3.9

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Abstract

Purpose: To express, purify and characterize recombinant mouse Cu/Zn-binding superoxide dismutase (mSOD1), and investigate its activity in vitro.Methods: The protein, mSOD1, was expressed after induction with isopropyl-beta-Dthiogalactopyranoside (IPTG). The target protein was purified by Ni-NTA affinity chromatography. The identity of the recombinant protein was confirmed by Western-blot and peptide mass fingerprinting(PMF) analysis. Protein activity in vitro was investigated by SOD activity assay kit and DNA damage protective assay.Results: mSOD1 protein was expressed with a final yield of about 60 mg of pure protein per liter of culture medium. After purification, the target protein was > 95 %. The identity of the recombinant protein was confirmed. SOD activity assay showed that the highest activity of the mSOD1 was 3789.0 ± 80.5 U/mg. The present work showed that mSOD1 was effective in protecting DNA from oxidative damage.Conclusion: High purity recombinant mSOD1 was obtained and characterized, and had high activity in vitro. The study indicates that the mSOD1 may serve as a potential therapeutic agent for those diseasescaused by oxidative stress.Keywords: Cu/Zn-binding Superoxide dismutase, Expression, Purification, Metal ions, DNA damage

Highlights

Superoxide dismutase (SOD; EC 1.15.1.1) is a ubiquitous enzyme, which catalyzes the dismutation of the superoxide radical into hydrogen peroxide and molecular oxygen (2O2+2H+ H2O2), and plays a central role in protecting against oxidative stress
The activity of mSOD1 in vivo has been related to amyotrophic lateral sclerosis [9,10], oxidative stress [11], reactive oxygen species (ROS) [12] and cerebral ischemia [13], most of these studies involved the use of SOD1 cDNA transfection
Integration of DNA fragment amplifier and sequence analysis indicated that it encoded a mature mouse Cu/Znbinding superoxide dismutase protein with an estimated molecular mass of 20 kDa

Summary

INTRODUCTION

Superoxide dismutase (SOD; EC 1.15.1.1) is a ubiquitous enzyme, which catalyzes the dismutation of the superoxide radical into hydrogen peroxide and molecular oxygen (2O2+2H+ H2O2), and plays a central role in protecting against oxidative stress. No data have been published on SOD1 sequences from mouse expressed with a modified pET-15b vector in E.coli Rosetta-gami cells. There has been no report on expression process optimization and productivity estimation of mouse SOD1. The pET15b-mSOD1 plasmid was transformed into E. coli Rosetta-gami strain for expression. A single colony of E. coli Rosetta-gami cells bearing pET15b-mSOD1 plasmid was inoculated into 5 ml LB medium containing 100 g/ml ampicillin. After filtering with a 0.22 m filter, the supernatant was loaded onto a Ni– NTA-His bind column equilibrated with buffer A (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.4) at 4 oC. The mSOD1 was eluted with buffer C (20 mM sodium phosphate, 500 mM NaCl, 250 mM imidazole, pH 7.4). The blocked membrane was washed twice for 5 min in TBST and incubated with mouse anti-His IgG (1:1000) for 2 h at 37 oC.

RESULTS

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CONCLUSION