Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones

Kaplan, Ondřej; Zárubová, Jana; Mikulová, Barbora; Filová, Elena; Bartova, Jirina; Bačáková, Lucie; Brynda, Eduard

doi:10.1371/journal.pone.0163697

Cited by 12 publications

(7 citation statements)

References 70 publications

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“…Disulfide bond formation is a critical step to obtain functional EGF growth factors. Therefore, many strategies have been attempted to overcome this problem, such as utilization of the E. coli Shuffle 30 and Origami 2 (DE3) 31 strains to try to control redox environment, secretion to the periplasm 32 , and fusion with chaperone-proteins, such as oleosin 33 , glutathione S-transferase 34 , SUMO 33 , B1 domain of streptococcal protein G 34 , protein disulfide isomerase (PDI) 35 , N-utilization substance protein A 35 , maltose-binding protein 35 , b'a' domain of PDI (PDIb'a') 35 , ELK16 36 and thioredoxin (Trx) 37 . In general, these studies have demonstrated the requirement of a fusion partner for proper folding of the EGF growth factors, which can be efficiently separated by proteolytic cleavage.…”

Section: Introductionmentioning

confidence: 99%

A toolkit for recombinant production of seven human EGF family growth factors in active conformation

Ferreira

Lopacinski

Batista

et al. 2022

Sci Rep

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Epidermal growth factors (EGF) play a wide range of roles in embryogenesis, skin development, immune response homeostasis. They are involved in several pathologies as well, including several cancer types, psoriasis, chronic pain and chronic kidney disease. All members share the structural EGF domain, which is responsible for receptor interaction, thereby initiating transduction of signals. EGF growth factors have intense use in fundamental research and high potential for biotechnological applications. However, due to their structural organization with three disulfide bonds, recombinant production of these factors in prokaryotic systems is not straightforward. A significant fraction usually forms inclusion bodies. For the fraction remaining soluble, misfolding and incomplete disulfide bond formation may affect the amount of active factor in solution, which can compromise experimental conclusions and biotechnological applications. In this work, we describe a reliable procedure to produce seven human growth factors of the EGF family in Escherichia coli. Biophysical and stability analyses using limited proteolysis, light scattering, circular dichroism and nanoDSF show that the recombinant factors present folded and stable conformation. Cell proliferation and scratch healing assays confirmed that the recombinant factors are highly active at concentrations as low as 5 ng/ml.

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Section: Introductionmentioning

confidence: 99%

A toolkit for recombinant production of seven human EGF family growth factors in active conformation

Ferreira

Lopacinski

Batista

et al. 2022

Sci Rep

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“…The reason we see a higher cell viability among TIB-HMVECs might be due to increased secretion of VEGF-A, figure 4(c). VEGF is a potent mitogen that regulates endothelial cell proliferation [28,29]. At 7 d, these VEGF values might equilibrate to those similar to MP-HMVECs that we see in figures 3(a) and (d).…”

Section: Effects Of Tib On Cell Viabilitymentioning

confidence: 68%

Thermal inkjet bioprinting triggers the activation of the VEGF pathway in human microvascular endothelial cells in vitro

et al. 2019

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One biofabrication process that has gained tremendous momentum in the field of tissue engineering and regenerative medicine is cell-printing or most commonly bioprinting. We have shown that thermal inkjet bioprinted human microvascular endothelial cells were recruited or otherwise involved in the formation of microvasculature to form graft-host anastomoses upon implantation. The present study aims to quantify and characterize the expression and activation of specific cytokines and kinases in vitro. Morphological characteristics demonstrate elongated protrusions of TIB-HMVECs at 5-6 times the size of manually pipetted cells. Moreover, annexin V-FITC and propidium iodide apoptosis assay via flow cytometry demonstrated a 75% apoptosis among printed cells as compared to among control cells. Cell viability at a 3 d incubation period was significantly higher for printed cells as compared to control. Milliplex magnetic bead panels confirmed significant overexpression of HSP70, IL-1α, VEGF-A, IL-8, and FGF-1 of printed cells compared to control. In addition, a Human phospho-kinase array displayed a significant over activation of the heat-shock proteins HSP27 and HSP60 of printed cells compared to the manually seeded cells. Collectively, it is suggested that the massive appearance of capillary blood vessels upon implantation that has been reported elsewhere may be due to the activation of the HSP-NF-κB pathway to produce VEGF. This cell activation may be used as a new strategy for vascularization of tissue engineered constructs which are in high demand in regenerative medicine applications.

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“…To overcome the mispairing of disulfide bonds, E. coli host strains have been genetically engineered to control the cellular redox environment [ 26 – 28 ]. As another strategy, solubility enhancers, such as maltose-binding protein (MBP) [ 1 , 17 ], oleosin [ 12 ], low-molecular-weight protamine [ 14 ], HaloTag [ 18 ], glutathione S -transferase [ 1 , 17 ], protein disulfide isomerase (PDI) [ 1 , 17 ], ELK16 [ 29 ], and thioredoxin [ 1 , 17 , 30 ], are fused to the target protein, including GFs. As the solubility of the tag affects the solubility of the passenger protein, choosing an appropriate solubility tag is critical [ 31 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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 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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Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones

Cited by 12 publications

References 70 publications

A toolkit for recombinant production of seven human EGF family growth factors in active conformation

A toolkit for recombinant production of seven human EGF family growth factors in active conformation

Thermal inkjet bioprinting triggers the activation of the VEGF pathway in human microvascular endothelial cells in vitro

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

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