scholarly journals Cloning, purification and enzymatic characterization of recombinant human Superoxide dismutase 1 expressed in Escherichia coli

2018 ◽  
Vol 65 (2) ◽  
pp. 235-240 ◽  
Author(s):  
LIN FENG ◽  
Yan Dan Dan ◽  
Chen Ya Wen ◽  
Fletcher Emmanuella E ◽  
Shi Hai Feng ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Expression Vector ◽  
Superoxide Dismutase 1 ◽  
Enzymatic Characterization ◽  
Activity Detection ◽  
Bivalent Cations ◽  
Bxpc3 Cell ◽  
Structure And Activity ◽  
Human Sod1

Superoxide dismutase 1 (SOD1) is a metalloenzyme that catalyzes disproportion-action superoxide into molecular oxygen and hydrogen peroxide. In this study, the human SOD1 (hSOD1) gene was cloned, expressed, and purified. The hSOD1 gene was amplified from a pool of Bxpc3 cell cDNAs by PCR and cloned into expression vector pET-28a (+). The recombinant soluble hSOD1 was expressed in E.coli BL21 (DE3) at 37°C and purified by Nickel column affinity chromatography. The soluble hSOD1 was produced with a yield of 5.9 ug/mL medium. Considering that metal ions have a certain influence on the structure and activity of protein, we researched the influences of different concentrations of Cu2+ and Zn2+ on hSOD1 activity at induction and the time of activity detection. The results implied Cu2+ and Zn2+ can’t enhance SOD1 expression, however can improve the catalytic activity at induction. Furthermore, most of bivalent cations have an improve effect on enzyme activity at the time of detection.

Combining in-cell NMR and X-ray fluorescence microscopy to reveal the intracellular maturation states of human superoxide dismutase 1

2015 ◽  
Vol 51 (3) ◽  
pp. 584-587 ◽  
Author(s):  
E. Luchinat ◽  
A. Gianoncelli ◽  
T. Mello ◽  
A. Galli ◽  
L. Banci
Keyword(s):  
Superoxide Dismutase ◽  
Nmr Spectroscopy ◽  
Fluorescence Microscopy ◽  
Superoxide Dismutase 1 ◽  
X Ray ◽  
Optical Fluorescence ◽  
Human Sod1

Combined in-cell NMR spectroscopy, X-ray fluorescence and optical fluorescence microscopies allow describing the intracellular maturation states of human SOD1.

Characterization of superoxide dismutase 1 (SOD-1) by electrospray ionization-ion mobility mass spectrometry

2013 ◽  
Vol 48 (1) ◽  
pp. 60-67 ◽  
Author(s):  
Marta Borges-Alvarez ◽  
Fernando Benavente ◽  
Marta Vilaseca ◽  
José Barbosa ◽  
Victoria Sanz-Nebot
Keyword(s):  
Mass Spectrometry ◽  
Superoxide Dismutase ◽  
Electrospray Ionization ◽  
Ion Mobility ◽  
Ion Mobility Mass Spectrometry ◽  
Superoxide Dismutase 1

Construction and Characterization of a Medium Copy Number Expression Vector Carrying Auto-Inducible dps Promoter to Overproduce a Bacterial Superoxide Dismutase in Escherichia coli

2019 ◽  
Vol 61 (4) ◽  
pp. 231-240 ◽  
Author(s):  
Debbie Soefie Retnoningrum ◽  
I. Wayan Martadi Santika ◽  
Suryanata Kesuma ◽  
Syahdu Ayu Ekowati ◽  
Catur Riani
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Copy Number ◽  
Expression Vector ◽  
Dps Promoter

scholarly journals Variation in the vulnerability of mice expressing human superoxide dismutase 1 to prion-like seeding: a study of the influence of primary amino acid sequence

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Jacob I. Ayers ◽  
Guilian Xu ◽  
Kristy Dillon ◽  
Qing Lu ◽  
Zhijuan Chen ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Motor Neuron ◽  
Motor Neuron Disease ◽  
Superoxide Dismutase 1 ◽  
Primary Sequence ◽  
Newborn Mice ◽  
Adult Mice ◽  
Ability To Act ◽  
Human Sod1

AbstractMisfolded forms of superoxide dismutase 1 (SOD1) with mutations associated with familial amyotrophic lateral sclerosis (fALS) exhibit prion characteristics, including the ability to act as seeds to accelerate motor neuron disease in mouse models. A key feature of infectious prion seeding is that the efficiency of transmission is governed by the primary sequence of prion protein (PrP). Isologous seeding, where the sequence of the PrP in the seed matches that of the host, is generally much more efficient than when there is a sequence mis-match. Here, we used paradigms in which mutant SOD1 seeding homogenates were injected intraspinally in newborn mice or into the sciatic nerve of adult mice, to assess the influence of SOD1 primary sequence on seeding efficiency. We observed a spectrum of seeding efficiencies depending upon both the SOD1 expressed by mice injected with seeds and the origin of the seed preparations. Mice expressing WT human SOD1 or the disease variant G37R were resistant to isologous seeding. Mice expressing G93A SOD1 were also largely resistant to isologous seeding, with limited success in one line of mice that express at low levels. By contrast, mice expressing human G85R-SOD1 were highly susceptible to isologous seeding but resistant to heterologous seeding by homogenates from paralyzed mice over-expressing mouse SOD1-G86R. In other seeding experiments with G85R SOD1:YFP mice, we observed that homogenates from paralyzed animals expressing the H46R or G37R variants of human SOD1 were less effective than seeds prepared from mice expressing the human G93A variant. These sequence mis-match effects were less pronounced when we used purified recombinant SOD1 that had been fibrilized in vitro as the seeding preparation. Collectively, our findings demonstrate diversity in the abilities of ALS variants of SOD1 to initiate or sustain prion-like propagation of misfolded conformations that produce motor neuron disease.

Safe and effective superoxide dismutase 1 silencing using artificial microRNA in macaques

2018 ◽  
Vol 10 (465) ◽  
pp. eaau6414 ◽  
Author(s):  
Florie Borel ◽  
Gwladys Gernoux ◽  
Huaming Sun ◽  
Rachel Stock ◽  
Meghan Blackwood ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Motor Neurons ◽  
Disease Onset ◽  
Mutant Gene ◽  
Artificial Microrna ◽  
Superoxide Dismutase 1 ◽  
Adeno Associated Virus ◽  
Efficient Delivery ◽  
Further Development

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease caused by degeneration of motor neurons leading to rapidly progressive paralysis. About 10% of cases are caused by gain-of-function mutations that are transmitted as dominant traits. A potential therapy for these cases is to suppress the expression of the mutant gene. Here, we investigated silencing of SOD1, a gene commonly mutated in familial ALS, using an adeno-associated virus (AAV) encoding an artificial microRNA (miRNA) that targeted SOD1. In a superoxide dismutase 1 (SOD1)–mediated mouse model of ALS, we have previously demonstrated that SOD1 silencing delayed disease onset, increased survival time, and reduced muscle loss and motor and respiratory impairments. Here, we describe the preclinical characterization of this approach in cynomolgus macaques (Macaca fascicularis) using an AAV serotype for delivery that has been shown to be safe in clinical trials. We optimized AAV delivery to the spinal cord by preimplantation of a catheter and placement of the subject with head down at 30° during intrathecal infusion. We compared different promoters for the expression of artificial miRNAs directed against mutant SOD1. Results demonstrated efficient delivery and effective silencing of the SOD1 gene in motor neurons. These results support the notion that gene therapy with an artificial miRNA targeting SOD1 is safe and merits further development for the treatment of mutant SOD1-linked ALS.

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