Enhanced secretion of biologically active, non-glycosylated VEGF from Saccharomyces cerevisiae

Kang, Woo Kyu; Lee, Min Hyung; Kim, Yeong Hyeock; Kim, Min Young; Kim, Jeong‐Yoon

doi:10.1016/j.jbiotec.2013.02.004

Cited by 8 publications

(5 citation statements)

References 28 publications

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“…VEGFs are synthesized and secreted by various cell types including endothelial cells, fibroblasts, macrophages, T cells, neutrophils, platelets, keratinocytes, renal mesangial cells, and many tumor cells [ 10 , 11 ]. Several strategies have been used to heterologously express and produce VEGFs in various eukaryotic expression host systems such as yeast [ 12 , 13 ], insect [ 14 ], rice [ 15 ], silkworm [ 16 ] and mammalian cells [ 17 ]. However, the production of VEGF in these systems can lead to impaired hyper-glycosylation and is limited by requiring strict cultivation conditions and specialized equipment.…”

Section: Introductionmentioning

confidence: 99%

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag

et al. 2016

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Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF). We created seven N-terminal fusion tag constructs with hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.

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Section: Introductionmentioning

confidence: 99%

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag

et al. 2016

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“…As an important example down regulation of total protein glycosylation by SR-BI depletion also affected STAT5, which reduced its binding to DNA as well as its target gene expression including VEGFA. Interestingly, intracellular transport and release of VEGFA from the cytoplasm strictly depends on glycosylation (37,38). Hence, loss of SR-BI not only reduces VEGFA mRNA expression, but additionally it prevents VEGFA maturation and secretion.…”

Section: Discussionmentioning

confidence: 99%

Malignant Phenotypes in Metastatic Melanoma are Governed by SR-BI and its Association with Glycosylation and STAT5 Activation

Kinslechner

Schörghofer

Schütz

et al. 2018

Molecular Cancer Research

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Metastatic melanoma is hallmarked by elevated glycolytic flux and alterations in cholesterol homeostasis. The contribution of cholesterol transporting receptors for the maintenance of a migratory and invasive phenotype is not well defined. Here, the scavenger receptor class B type I (SCARB1/SR-BI), a high-density lipoprotein (HDL) receptor, was identified as an estimator of melanoma progression in patients. We further aimed to identify the SR-BI-controlled gene expression signature and its related cellular phenotypes. On the basis of whole transcriptome analysis, it was found that SR-BI knockdown, but not functional inhibition of its cholesterol-transporting capacity, perturbed the metastasis-associated epithelial-to-mesenchymal transition (EMT) phenotype. Furthermore, SR-BI knockdown was accompanied by decreased migration and invasion of melanoma cells and reduced xenograft tumor growth. STAT5 is an important mediator of the EMT process and loss of SR-BI resulted in decreased glycosylation, reduced DNA binding, and target gene expression of STAT5. When human metastatic melanoma clinical specimens were analyzed for the abundance of SR-BI and STAT5 protein, a positive correlation was found. Finally, a novel SR-BI-regulated gene profile was determined, which discriminates metastatic from nonmetastatic melanoma specimens indicating that SR-BI drives gene expression contributing to growth at metastatic sites. Overall, these results demonstrate that SR-BI is a highly expressed receptor in human metastatic melanoma and is crucial for the maintenance of the metastatic phenotype. High SR-BI expression in melanoma is linked with increased cellular glycosylation and hence is essential for a metastasis-specific expression signature. .

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“…The highest expression level of the analysed clones was 5.7 mg/L of VEGF 165 . It has been reported that recombinant human VEGF 165 is expressed in various expression systems, such as E. coli at 1.5 mg/L (Taktak-BenAmar et al 2017) and Saccharomyces cerevisiae at 4 mg/L (Kang et al 2013). In other studies, the expression levels of 80 mg/L (Lee et al 2008) for CHO cell cultures and 20 mg/L (Lee et al 2006) for insect cells at were achieved.…”

Section: Expression Of Vegf 165 In K Lactismentioning

confidence: 99%

“…VEGF is synthesized by numerous cell types, including endothelial cells, tumour cells, broblasts, platelets, macrophages, neutrophils, keratinocytes, T cells and renal mesangial cells (Boocock et al 1995;Sunderkotter et al 1994, Verheul et al 1997Frank et al 1995;Iijima et al 1993). Aside from bacterial expression (Pizarro et al 2010;Nguyen et al 2016), several eukaryotic expression host systems have been employed for the recombinant production of VEGF, including yeast (Mohanraj et al 1995;Kang et al 2013), Chinese hamster ovary cells (CHOs) (Lee et al 2008), insect cells (Lee et al 2006), transgenic rice (Chung et al 2014) and silkworm (Wu et al 2004). The most preferred system is Escherichia coli (E. coli); however, it exhibits drawbacks, such as the tendency to form inclusion bodies, protein misfolding and di cult puri cation steps (Nguyen et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Expression and characterization of functional human vascular endothelial growth factor (VEGF165) in Kluyveromyces lactis

Kuduğ

Tayhan

Gökçe

2020

Preprint

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Among the vascular endothelial growth factor (VEGF) family variants, the 165-amino acid isoform (VEGF165) is the best characterized and most potent endothelial cell mitogenic factor. It is known that VEGF165 mediates angiogenesis and has the potential for the therapeutic applications. In this study, the expression system of Kluyveromyces lactis that produces the recombinant human VEGF165 has been evaluated. The gene encoding human VEGF165 was successfully cloned in the pKLAC2 expression vector containing a strong LAC4 promoter, after which a pKLAC2-VEGF165 plasmid was constructed. After the transformation, the recombinant human vascular endothelial growth factor 165 (rhVEGF165) was expressed in K. lactis GG799 cells (~ 5.7 mg/L) confirmed by SDS-PAGE and Western blotting and downstream purification processed comprising ammonium sulphate precipitation and affinity chromatography. The biological activity of the purified rhVEGF165 was confirmed by the proliferation of the human umbilical vein-derived endothelial cells (HUVEC) in a dose- and time-dependent manner. The K. lactis-derived rhVEGF165 exhibited a higher proliferative activity compared with a commercially available rhVEGF165 with a half-maximal effective concentration of 3.0. Cell migration analysis was conducted to evaluate the in vitro wound healing effect of the produced rhVEGF165 via a scratch assay. These findings indicate that K. lactis could be a suitable host for secreting bioactive human VEGF165 for therapeutic use.

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Enhanced secretion of biologically active, non-glycosylated VEGF from Saccharomyces cerevisiae

Cited by 8 publications

References 28 publications

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag

Malignant Phenotypes in Metastatic Melanoma are Governed by SR-BI and its Association with Glycosylation and STAT5 Activation

Expression and characterization of functional human vascular endothelial growth factor (VEGF165) in Kluyveromyces lactis

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