High-level production of N-terminal pro-brain natriuretic peptide, as a calibrant of heart failure diagnosis, in Escherichia coli

Kim, Young Su; Karisa, Nadia; Jeon, Woo Young; Lee, Hongweon; Kim, Yeu-chun; Ahn, Jungoh

doi:10.1007/s00253-019-09826-8

Cited by 12 publications

(14 citation statements)

References 28 publications

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“…S2a and b). We therefore optimized the TEV protease treatment conditions by using additives such as β-mercaptoethanol, Tween-20, and Triton X-100 [16,37,[46][47][48][49]. In the case of aFGF, almost half the aFGF and fused aFGF aggregated after treatment with 5% (w/w) TEV protease in 1⋅ phosphate-buffered saline (PBS) supplemented with 150 mM NaCl (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Costa et al [32,35,36] demonstrated that Fh8, an 8-kDa protein, and Fh8 with an N-terminal hexahistidine (6HFh8) could be used as a fusion partner that increases the solubility of various target proteins to a greater extent than other protein tags. Further, we showed that using 6HFh8 as a fusion partner facilitates the solubilization of N-terminal pro-brain natriuretic peptide, which increases approximately 97.5-fold in the nal product yield [37].…”

Section: Introductionmentioning

confidence: 90%

“…It was fused at the N-terminus of GFs via a stable linker peptide S 5 N 10 ( Fig. 1) [37]. Further, hexa-histidine (6H) was attached to the N-terminus of the Fh8 tag (6HFh8) to facilitate the puri cation of fusion proteins.…”

Section: Construction Of An Expression Vector For Human Gfs Fused To Fh8mentioning

confidence: 99%

“…Genes encoding the GFs with 6HFh8 were codon-optimized and synthesized by DNA 2.0 (ATUM, Menlo Park, CA, USA). The fusion proteins were constructed as following our previous report [37]. Brie y, hexahistidine fused Fh8 (6HFh8) was fused to N-terminus of GFs via the S 5 N 10 linker and TEV protease cleavage site (ENLYFQ-G/S) in the P1′ position.…”

Section: Construction Of Protein Expression Vectors and Strainsmentioning

confidence: 99%

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Efficient production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

Kim

Lee

Han

et al. 2020

Preprint

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Background: Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms.Results: We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein on a g/l scale. The two GFs were successfully highly purified (>99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents.Conclusions: We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 90%

Section: Construction Of An Expression Vector For Human Gfs Fused To Fh8mentioning

confidence: 99%

Section: Construction Of Protein Expression Vectors and Strainsmentioning

confidence: 99%

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Efficient production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

Kim

Lee

Han

et al. 2020

Preprint

Self Cite

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“…It was fused at the N-terminus of GFs via a stable linker peptide S 5 N 10 ( Fig. 1) [36]. Further, hexa-histidine (6H) was attached to the N-terminus of the Fh8 tag (6HFh8) to facilitate the purification of fusion proteins.…”

Section: Construction Of An Expression Vector For Human Gfs Fused To Fh8mentioning

confidence: 99%

Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

et al. 2021

Self Cite

View full text Add to dashboard Cite

Background Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms. Results We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents. Conclusions We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

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Tagging Recombinant Proteins to Enhance Solubility and Aid Purification

Loughran,

Walls

2023

Methods in Molecular Biology

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High-level production of N-terminal pro-brain natriuretic peptide, as a calibrant of heart failure diagnosis, in Escherichia coli

Cited by 12 publications

References 28 publications

Efficient production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

Efficient production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

Tagging Recombinant Proteins to Enhance Solubility and Aid Purification

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