Production of functional human vascular endothelial growth factor165 in transgenic rice cell suspension cultures

Chung, Nguyen-Duc; Kim, Nan-Sun; Giap, Do Van; Jang, Seon-Hui; Oh, Sun-Mi; Jang, Seong Soon; Kim, Tae-Geum; Jang, Yong-Suk; Yang, Moon-Sik

doi:10.1016/j.enzmictec.2014.05.007

Cited by 22 publications

(11 citation statements)

References 37 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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“…Rice cell suspension cultures may overcome the problem of low expression levels by using the rice α-amylase 3D promoter system. The α-amylase 3D promoter, which is activated by sugar starvation [66], is one of the most widely used metabolite-regulated promoters; indeed, it is used in rice cell suspension cultures to produce various recombinant proteins such as hGM-CSF [41], human growth hormone [42], human VEGF165 [43], FimA mAb [44], bovine trypsin [38], and human pepsinogen C [45].…”

Section: Cell Cultures In "Stable" Systemsmentioning

confidence: 99%

Development of Systems for the Production of Plant-Derived Biopharmaceuticals

Moon

Park

et al. 2019

Plants

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Over the last several decades, plants have been developed as a platform for the production of useful recombinant proteins due to a number of advantages, including rapid production and scalability, the ability to produce unique glycoforms, and the intrinsic safety of food crops. The expression methods used to produce target proteins are divided into stable and transient systems depending on applications that use whole plants or minimally processed forms. In the early stages of research, stable expression systems were mostly used; however, in recent years, transient expression systems have been preferred. The production of the plant itself, which produces recombinant proteins, is currently divided into two major approaches, open-field cultivation and closed-indoor systems. The latter encompasses such regimes as greenhouses, vertical farming units, cell bioreactors, and hydroponic systems. Various aspects of each system will be discussed in this review, which focuses mainly on practical examples and commercially feasible approaches.

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“…In addition, growth rate in rice cell suspension culture is relatively high with cell doubling time of 1.5-1.7 days, spherical cell structure, and minimal cell aggregation in comparison to other plant cell cultures (Santos et al, 2016;Trexler et al, 2002b). Utilization of rice cell suspension culture and Ramy3D expression system have resulted in e cient and high-level expression of many recombinant proteins including human serum albumin (Huang et al, 2005;Liu et al, 2015), human lysozyme (Huang et al, 2002), hVEGF 165 (Chung et al, 2014), bovine trypsin (Kim et al, 2011) BFGF is also well-known in studies related to stem cell because it maintains the undifferentiated and pluripotent state of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and is one of the essential components of their cultures (Ding et al, 2010;Levenstein et al, 2006). The hESCs and hiPSCs cells are able to differentiate into all cell types of the three germ layers (Smith and biology, 2001).…”

Section: Introductionmentioning

confidence: 99%

Human basic Fibroblastic Growth Factor production by codon optimization in rice cell suspension culture

bastami¹,

Hosseini²

2022

Preprint

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Human basic fibroblastic growth factor (hbFGF), possesses a wide range of highly important biological functions, including cell proliferation, protein synthesis and metabolism. This study is the first investigation to transfer a partly home-made hbFGF gene cassette into rice cells. The human bFGF coding sequence was optimized for expression in rice in order to improve its in vitro production. Following synthesis, the expression cassette was constructed by fusing the PCR- amplified Ram3D promoter and terminator sequences in two separate steps to the optimized hbFGF CDS. Then, the cassette was transferred into the pCAMBIA1304 shuttle vector. This vector also contained a signal peptide for the secretion of the recombinant protein into the culture medium. Rice (Oryza sativa cv. Kanto 51) seeds were cultured on the 2N6PG medium composing of the N6 basal medium supplemented with 2 mg/L 2,4-D for callus induction. Callus transformation was performed by employing Agrobacterium tumefaciens strain LBA 4404. The integration of bFGF gene into the chromosomes of transgenic calli was verified by genomic DNA PCR. A transformed callus line expressing the highest level of bFGF (38.1 mg/kg FW) was recognized by ELISA and selected for the establishment of cell suspension culture. The developed transgenic rice cell culture was able to express and secrete the recombinant bFGF into the culture medium on day 3 after incubation of cells in the sucrose-free medium. The SDS-PAGE and Western blot analyses detected the bFGF in the culture medium with a molecular weight of ~17 kDa under reducing conditions. The purification of bFGF was performed using HiTrap Heparin HP column. Interestingly, the activity assay indicated that the rice derived bFGF stimulated the proliferation of NIH/3T3 cells similar to the commercial bFGF.

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Production of functional human vascular endothelial growth factor165 in transgenic rice cell suspension cultures

Cited by 22 publications

References 37 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

Development of Systems for the Production of Plant-Derived Biopharmaceuticals

Human basic Fibroblastic Growth Factor production by codon optimization in rice cell suspension culture

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