High Efficient Expression of Bioactive Human BMP-14 in E. coli Using SUMO Fusion Partner

Li, Jian Feng; Cui, Xian Wei; Ji, Hai Yan; Qiu, Ting; Ji, Xue; Du, Ming Xian; Wu, Hai; Xu, Xing; Zhang, S. Q.

doi:10.1007/s10930-011-9368-3

Cited by 6 publications

(1 citation statement)

References 15 publications

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“…The SUMO protease recognizes the SUMO fusion partner, facilitating precise proteolytic cleavage, thus resulting in the production of native proteins. pET SUMO has been widely used to produce many therapeutically important proteins, including human interferon alpha-2a (Bis et al 2014 ), BMP-14 (Li et al 2011 ), human interleukin-11 (Nguyen et al 2018 ), and FGF-21 (Wang et al 2010 ).…”

Section: Discussionmentioning

confidence: 99%

Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system

et al. 2023

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Current research focuses on the soluble and high-level expression of biologically active recombinant human IL-29 protein in Escherichia coli . The codon-optimized IL-29 gene was cloned into the Champion™ pET SUMO expression system downstream of the SUMO tag under the influence of the T7 lac promoter. The expression of SUMO-fused IL-29 protein was compared in E. coli Rosetta 2(DE3), Rosetta 2(DE3) pLysS, and Rosetta-gami 2(DE3). The release of the SUMO fusion partner resulted in approximately 98 mg of native rhIL-29 protein with a purity of 99% from 1 l of fermentation culture. Purified rhIL-29 was found to be biologically active, as evaluated by its anti-proliferation assay. It was found that Champion™ pET SUMO expression system can be used to obtained high yield of biologically active soluble recombinant human protein compared to other expression vector.

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Section: Discussionmentioning

confidence: 99%

Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system

et al. 2023

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Small Ubiquitin-Like Modifier Protein 3 Enhances the Solubilization of Human Bone Morphogenetic Protein 2 in E. coli

Hanif

Yaseen

Gul

et al. 2018

Appl Biochem Biotechnol

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Small ubiquitin-like modifier (SUMO) fusion technology is widely used in the production of heterologous proteins from prokaryotic system to aid in protein solubilization and refolding. Due to an extensive clinical application of human bone morphogenetic protein 2 (hBMP2) in bone augmentation, total RNA was isolated from human gingival tissue and mature gene was amplified through RT-PCR, cloned (pET21a), sequence analyzed, and submitted to GenBank (Accession no. KF250425). To obtain soluble expression, SUMO3 was tagged at the N-terminus of hBMP2 gene (pET21a/SUMO3-hBMP2), transferred in BL21 codon+, and ~ 40% soluble expression was obtained on induction with IPTG. The dimerized hBMP2 was confirmed with Western blot, native PAGE analysis, and purified by fast protein liquid chromatography with 0.5 M NaCl elution. The cleavage of SUMO3 tag from hBMP2 converted it to an insoluble form. Computational 3D structural analysis of the SUMO3-hBMP2 was performed and optimized by molecular dynamic simulation. Protein-protein interaction of SUMO3-hBMP2 with BMP2 receptor was carried out using HADDOCK and inferred stable interaction. The alkaline phosphatase assay of SUMO3-hBMP2 on C2C12 cells showed maximum 200-ng/ml dose-dependent activity. We conclude that SUMO3-tagged hBMP2 is more suited for generation of soluble form of the protein and addition of SUMO3 tag does not affect the functional activity of hBMP2.

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Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in Escherichia coli

et al. 2018

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Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon + and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~ 25.5% and ~ 6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.

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High Efficient Expression of Bioactive Human BMP-14 in E. coli Using SUMO Fusion Partner

Cited by 6 publications

References 15 publications

Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system

Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system

Small Ubiquitin-Like Modifier Protein 3 Enhances the Solubilization of Human Bone Morphogenetic Protein 2 in E. coli

Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in Escherichia coli

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