Expression analysis and ATR-FTIR characterization of the secondary structure of recombinant human TNF-α from Escherichia coli SHuffle ® T7 Express and BL21 (DE3) cells

Safarpour, Hossein; Banadkoki, Sahar Barzegari; Keshavarzi, Zahra; Morowvat, Mohammad Hossein; Soleimanpour, Mahdieh; Pourmolaei, Shokoufeh; Shirazi, Farshad H.

doi:10.1016/j.ijbiomac.2017.02.052

Cited by 22 publications

(9 citation statements)

References 27 publications

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“…As SHuffle ® cells are under oxidative stress, high metabolic activity during growth at high temperatures such as 37 °C may have detrimental effects on its or growth (Lobstein et al 2012). Consistent with the results of the present study, other studies including our previous study reported (Ahmadzadeh et al 2020) 30 °C as proper induction temperature for recombinant protein expression in SHuffle ® strain (Safarpour et al 2017).…”

Section: Discussionsupporting

confidence: 90%

Cloning, Expression and One-Step Purification of a Novel IP-10-(anti-HER2 scFv) Fusion Protein in Escherichia coli

Ahmadzadeh

Farshdari

Behdani

et al. 2020

Int J Pept Res Ther

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Breast cancer is the most common cancer in women, worldwide. The correlation between breast cancer malignancy and human epidermal growth factor 2 (HER2) expression leads to application of monoclonal antibodies against HER2 in HER2overexpressing breast cancers. Variable fragments of light and heavy chains of monoclonal antibodies are linked in single chain variable fragment (scFv). Herein, IP-10 chemokine is conjugated to scFv against HER2 and thus CD8 + T cells can chemoattract to HER2-overexpressing tumors. IP-10-(anti-HER2 scFv) gene was cloned in pET22-b (+) and successful expression of the fusion protein in E. coli host was confirmed by detecting a 39 kDa band in Western blotting. The highest fusion protein expression in Origami (DE3), BL21 (DE3) and SHuffle ® were obtained 4 h after 0.1, 0.5 and 0.1 mM IPTG induction at 37, 37 and 30 °C, respectively. It was also demonstrated that the most solubility of the fusion protein is obtained at 25 °C in all the three examined strains. The highest soluble form of the fusion protein was expressed in SHuffle ® . However, due to low amount of the fusion protein expressed in SHuffle ® , we focused on its expression in Origami (DE3). Finally, purification of the fusion protein using Ni-NTA affinity chromatography under native, denaturing and hybrid conditions was investigated. Purification under hybrid condition caused the most purity of the fusion protein. This study opens up the avenue of exploring the use of IP-10-(anti-HER2 scFv) as a potential treatment for HER2-overexpresing tumors or as an adjuvant in combination with HER2-based vaccine.

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Section: Discussionsupporting

confidence: 90%

Cloning, Expression and One-Step Purification of a Novel IP-10-(anti-HER2 scFv) Fusion Protein in Escherichia coli

Ahmadzadeh

Farshdari

Behdani

et al. 2020

Int J Pept Res Ther

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“…compared the expression of tumor necrosis factor alpha (TNF-a), a protein containing one disulfide bond, in BL21 (DE3) and SHuffle® T7 ( 27 ). Despite the higher yield of protein expression in BL21 (DE3), 1.5 fold higher disulfide bind formation was observed in TNF-a expressed in Shuffle T7 ( 27 ). It may be attributed to more oxidative cytoplasm of the SHuffle® T7 strain.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains

2020

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Background and purpose: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with a wide range of therapeutic applications although expression of GM-CSF in Escherichia coli ( E. coli ) usually leads to formation of insoluble aggregates mostly lack biological activity. The aim of this study was to compare the soluble expression level of GM-CSF in three E. coli strains, BL21 (DE3), SHuffle® T7 and Origami™ 2 (DE3). Experimental approach: The effect of different temperatures and inducer concentrations on soluble expression of GM-CSF was evaluated. The soluble GM-CSF was subjected to endotoxin removal and purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, ultrafiltration. The biological activity of produced GM-CSF was evaluated based on its growth promotion effect on TF-1 cell lines by MTT assay method. Findings / Results: A significant improvement of the soluble yield of GM-CSF (about 30% of GM-CSF was expressed as soluble proteins) was observed when protein expression was induced at 30 °C with 0.5 mM isopropyl β- d-1-thiogalactopyranoside (IPTG) in E. coli Shuffle T7. The soluble GM-CSF with a high purity up to 95 % and specific activity of 1.25 × 10 4 IU/μg was obtained. Conclusion and implications: The proposed strategy here can be used to improve the soluble expression of other hard-to-express proteins with similar structural properties ( i.e ., containing disulfide binds or cysteine).

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“…The expression of the recombinant β-Glu in E.coli JM109 (DE3), cell harvest, and purification of protein were performed as described for the Tm-β-Glu-Tt-ChBD and Tm-β-Glu 7 . SDS-PAGE analysis was used to detect the molecular mass, purity, and heat treatment of Tm-β-Glu 37 . The molecular protein weight markers 100 kDa (PageRuler Prestained Protein Ladder, Thermo scientific, 26617) were used in this work.…”

Section: Methodsmentioning

confidence: 99%

Immobilization of β-Glucosidase from Thermatoga maritima on Chitin-functionalized Magnetic Nanoparticle via a Novel Thermostable Chitin-binding Domain

Alnadari

Xue

Zhou

et al. 2020

Sci Rep

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Enzyme immobilization is a powerful tool not only as a protective agent against harsh reaction conditions but also for the enhancement of enzyme activity, stability, reusability, and for the improvement of enzyme properties as well. Herein, immobilization of β-glucosidase from Thermotoga maritima (tm-β-Glu) on magnetic nanoparticles (Mnps) functionalized with chitin (ch) was investigated. This technology showed a novel thermostable chitin-binding domain (Tt-ChBD), which is more desirable in a wide range of large-scale applications. This exclusive approach was fabricated to improve the Galacto-oligosaccharide (GOS) production from a cheap and abundant by-product such as lactose through a novel green synthesis route. Additionally, SDS-PAGE, enzyme activity kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) revealed that among the immobilization strategies for Thermotoga maritime-β-Glucosidase thermostable chitinbinding domain (tm-β-Glu-Tt-ChBD) on the attractive substrate; Ch-MNPs had the highest enzyme binding capacity and GOS production ratio when compared to the native enzyme. More interestingly, a magnetic separation technique was successfully employed in recycling the immobilized Tm-β-Glu for repetitive batch-wise GOS without significant loss or reduction of enzyme activity. This immobilization system displayed an operative stability status under various parameters, for instance, temperature, pH, thermal conditions, storage stabilities, and enzyme kinetics when compared with the native enzyme. Conclusively, the GOS yield and residual activity of the immobilized enzyme after the 10 th cycles were 31.23% and 66%, respectively. Whereas the GOS yield from native enzyme synthesis was just 25% after 12 h in the first batch. This study recommends applying Tt-ChBD in the immobilization process of tm-β-Glu on Ch-MNPs to produce a low-cost GOS as a new eco-friendly process besides increasing the biostability and efficiency of the immobilized enzyme.

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Expression analysis and ATR-FTIR characterization of the secondary structure of recombinant human TNF-α from Escherichia coli SHuffle ® T7 Express and BL21 (DE3) cells

Cited by 22 publications

References 27 publications

Cloning, Expression and One-Step Purification of a Novel IP-10-(anti-HER2 scFv) Fusion Protein in Escherichia coli

Cloning, Expression and One-Step Purification of a Novel IP-10-(anti-HER2 scFv) Fusion Protein in Escherichia coli

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains

Immobilization of β-Glucosidase from Thermatoga maritima on Chitin-functionalized Magnetic Nanoparticle via a Novel Thermostable Chitin-binding Domain

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