Dataset Information

Enhancement of Solubility and Specific Activity of a Cu/Zn Superoxide Dismutase by Co-expression with a Copper Chaperone in Escherichia coli.

ABSTRACT: BACKGROUND:Human Cu/Zn superoxide dismutase (hSOD1) is an antioxidant enzyme with potential as a therapeutic agent. However, heterologous expression of hSOD1 has remained an issue due to Cu2+ insufficiency at protein active site, leading to low solubility and enzymatic activity. OBJECTIVES:The effect of co-expressed human copper chaperone (hCCS) to enhance the solubility and enzymatic activity of hSOD1 in E. coli was investigated in the presence and absence of Cu2+. MATERIALS AND METHODS:pETDuet-1-hSOD1 and pETDuet-1-hCCS-hSOD1 were constructed and individually transformed into E. coli strain BL21(DE3). The recombinant hSOD1 was expressed and purified using immobilized metal affinity chromatography. The yield and specific activity of hSOD1 in all conditions were studied. RESULTS:Co-expression with hCCS increased hSOD1 solubility at 37°C, but this effect was not observed at 25°C. Notably, the specific activity of hSOD1 was enhanced by 1.5 fold and greater than 3 fold when co-expressed with hCCS at 25°C with and without Cu2+ supplement, respectively. However, the chaperone co-expression did not significantly increase the yield of hSOD1 comparable to the expression of hSOD1 alone. CONCLUSIONS:This study is the first report demonstrating a potential use of hCCS for heterologous production of hSOD1 with high enzymatic activity.

SUBMITTER: Eiamphungporn W

PROVIDER: S-EPMC5434994 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Enhancement of Solubility and Specific Activity of a Cu/Zn Superoxide Dismutase by Co-expression with a Copper Chaperone in <i>Escherichia coli</i>.

Eiamphungporn Warawan W Yainoy Sakda S Prachayasittikul Virapong V

Iranian journal of biotechnology 20161201 4

<h4>Background</h4>Human Cu/Zn superoxide dismutase (hSOD1) is an antioxidant enzyme with potential as a therapeutic agent. However, heterologous expression of hSOD1 has remained an issue due to Cu<sup>2+</sup> insufficiency at protein active site, leading to low solubility and enzymatic activity.<h4>Objectives</h4>The effect of co-expressed human copper chaperone (hCCS) to enhance the solubility and enzymatic activity of hSOD1 in <i>E. coli</i> was investigated in the presence and absence of Cu ...[more]

PMID: 28959342

Similar Datasets

Project description:Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme's activation is independent of a chaperone both for disulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts.Mimiviridae are giant viruses encoding more than 1,000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole's oxidative stress. The megavirus chilensis virion contains a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some megavirus chilensis relatives but is absent from mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the features that it has in common with and its differences from cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon supplementation of the growth medium with Cu and Zn, the recombinant protein is fully active, suggesting that the virus's SOD activation is independent of a copper chaperone for SOD generally used by eukaryotic SODs.

Project description:BackgroundCopper oxide (CuO) nanoparticles (NPs) are known to trigger cytotoxicity in a variety of cell models, but the mechanism of cell death remains unknown. Here we addressed the mechanism of cytotoxicity in macrophages exposed to CuO NPs versus copper chloride (CuCl2).MethodsThe mouse macrophage cell line RAW264.7 was used as an in vitro model. Particle uptake and the cellular dose of Cu were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. The deposition of Cu in lysosomes isolated from macrophages was also determined by ICP-MS. Cell viability (metabolic activity) was assessed using the Alamar Blue assay, and oxidative stress was monitored by a variety of methods including a luminescence-based assay for cellular glutathione (GSH), and flow cytometry-based detection of mitochondrial superoxide and mitochondrial membrane potential. Protein aggregation was determined by confocal microscopy using an aggresome-specific dye and protein misfolding was determined by circular dichroism (CD) spectroscopy. Lastly, proteasome activity was investigated using a fluorometric assay.ResultsWe observed rapid cellular uptake of CuO NPs in macrophages with deposition in lysosomes. CuO NP-elicited cell death was characterized by mitochondrial swelling with signs of oxidative stress including the production of mitochondrial superoxide and cellular depletion of GSH. We also observed a dose-dependent accumulation of polyubiquitinated proteins and loss of proteasomal function in CuO NP-exposed cells, and we could demonstrate misfolding and mitochondrial translocation of superoxide dismutase 1 (SOD1), a Cu/Zn-dependent enzyme that plays a pivotal role in the defense against oxidative stress. The chelation of copper ions using tetrathiomolybdate (TTM) prevented cell death whereas inhibition of the cellular SOD1 chaperone aggravated toxicity. Moreover, CuO NP-triggered cell death was insensitive to the pan-caspase inhibitor, zVAD-fmk, and to wortmannin, an inhibitor of autophagy, implying that this was a non-apoptotic cell death. ZnO NPs, on the other hand, triggered autophagic cell death.ConclusionsCuO NPs undergo dissolution in lysosomes leading to copper-dependent macrophage cell death characterized by protein misfolding and proteasomal insufficiency. Specifically, we present novel evidence for Cu-induced SOD1 misfolding which accords with the pronounced oxidative stress observed in CuO NP-exposed macrophages. These results are relevant for our understanding of the consequences of inadvertent human exposure to CuO NPs.

Dataset Information

Enhancement of Solubility and Specific Activity of a Cu/Zn Superoxide Dismutase by Co-expression with a Copper Chaperone in Escherichia coli.

Publications

Enhancement of Solubility and Specific Activity of a Cu/Zn Superoxide Dismutase by Co-expression with a Copper Chaperone in <i>Escherichia coli</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets