Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures

Kim, Nan-Sun; Yu, Houqing; Chung, Nguyen-Duc; Shin, Yun-Ji; Kwon, Taeho; Yang, Moon-Sik

doi:10.1016/j.pep.2010.10.007

Cited by 31 publications

(7 citation statements)

References 41 publications

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“…The advantage of this approach is that many different foreign domains may be fused to a target protein to increase its expression level or solubility and then be removed after expression in vivo, thereby allowing production of a target protein without any extra foreign domains. This strategy has been widely used for the production of many endogenous proteins, including growth factors and cytokines in animal cells as well as proteases such as pepsin and trypsin (Shi et al, 2011;Kim et al, 2011;Shen et al, 2017). These proteins are expressed as preproproteins and then converted to their functional form via proteolytic processing.…”

Section: Discussionmentioning

confidence: 99%

In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana

et al. 2020

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Plants show great potential for producing recombinant proteins in a cost-effective manner. Many strategies have therefore been employed to express high levels of recombinant proteins in plants. Although foreign domains are fused to target proteins for high expression or as an affinity tag for purification, the retention of foreign domains on a target protein may be undesirable, especially for biomedical purposes. Thus, their removal is often crucial at a certain time point after translation. Here, we developed a new strategy to produce target proteins without foreign domains. This involved in vivo removal of foreign domains fused to the N-terminus by the small ubiquitin-related modifier (SUMO) domain/SUMO-specific protease system. This strategy was tested successfully by generating a recombinant gene, BiP:p38:bdSUMO : His:hLIF, that produced human leukemia inhibitory factor (hLIF) fused to p38, a coat protein of the Turnip crinkle virus; the inclusion of p38 increased levels of protein expression. The recombinant protein was expressed at high levels in the leaf tissue of Nicotiana benthamiana. Coexpression of bdSENP1, a SUMO-specific protease, proteolytically released His:hLIF from the full-length recombinant protein in the endoplasmic reticulum of N. benthamiana leaf cells. His:hLIF was purified from leaf extracts via Ni 2+-NTA affinity purification resulting in a yield of 32.49 mg/kg, and the Nterminal 5-residues were verified by amino acid sequencing. Plant-produced His:hLIF was able to maintain the pluripotency of mouse embryonic stem cells. This technique thus provides a novel method of removing foreign domains from a target protein in planta.

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Section: Discussionmentioning

confidence: 99%

In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana

et al. 2020

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“…The ER signal peptide facilitated expression and secretion of intracellular rhEPO from hairy root cultures of tobacco (N. tabacum), resulting in a yield of up to 66.75 ng/g TSP [36]. In many cases [37][38][39], the promoter and signal peptide from the rice α-amylase gene have been used to develop a two-step process (increasing cell number and maintaining cell viability/activity in the first step and then producing recombinant protein in the second step), resulting in high production of secreted proteins. Although this system has some strengths, it increases both the cost of the process (as it is more labor-intensive) and the risk of contamination when changing the medium.…”

Section: Cell Cultures In "Stable" Systemsmentioning

confidence: 99%

“…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 recombinant bovine trypsin in transgenic rice cell suspension cultures

Cited by 31 publications

References 41 publications

In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana

In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana

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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