Production of mouse granulocyte‐macrophage colony‐stimulating factor by gateway technology and transgenic rice cell culture

Abstract

To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.

Therapeutically Important Proteins From In Vitro Plant Tissue Culture Systems

“…Tobacco cell lines such as BY-2 (bright yellow 2) and NT-1 (Nicotiana tabacum 1) are readily available, are amenable to Agrobacteriummediated genetic transformation, and have been optimised for rapid cell growth. Other host species applied for in vitro foreign protein synthesis include rice [24], soybean [25], tomato [26], carrot [18,27], Arabidopsis [28], Medicago truncatula [28] and N. benthamiana [29]. When in vitro plant cultures are used for protein production, the growth habit of the host species, such as whether it grows upright, is a high biomass producer in agronomic terms, or is suitable for mechanised agriculture, is no longer an important consideration.…”

Production of Recombinant Anti-Cancer Vaccines in Plants

“…These suspension cultures can even secrete recombinant proteins out of the cell into the culture medium when the signal peptide is fused to the proteins, making the cell lysis step unnecessary for extraction (Liu et al ., 2012). Carrot cells have been used for in vitro suspension-based production of diverse recombinant therapeutic proteins (Shaaltiel et al ., 2007), building on the successful establishment of a carrot cell suspension culture in 1970 (Nishi and Sugano, 1970).…”

Improving Pharmaceutical Protein Production in Oryza sativa

“…On the other hand, suspension cell cultures also have comparative advantages such as more controlled environment, faster cell growth, and potential to be upscaled into bioreactors [17]. In addition, by utilizing a signal peptide, recombinant proteins produced will be secreted out of the cell into the culture medium, removing the need to extract the protein through cell lysis [25]. Rice has been successfully used to produce human serum albumin, vaccine, antibodies, cytokines, and other pharmaceutical proteins [26].…”

“…The maximum yields were 1.3% of total soluble protein in rice seeds and 0.03% of total soluble protein in tobacco seeds [31,62]. Mouse GM-CSF (mGM-CSF) was successfully produced using rice suspension cells, with yield of 24.6 mg/L of medium [25]. The same glycoprotein was also produced in tobacco leaves, and the yield was 19 μg/g of fresh leaves [37].…”

“…The same glycoprotein was also produced in tobacco leaves, and the yield was 19 μg/g of fresh leaves [37]. The biological activities of recombinant human and mouse GM-CSF produced in rice suspension culture has also been confirmed using GM-CSF dependent TF1 cells and murine myeloblastic leukemia NFS-60 cells, respectively [8,25]. …”