Biologically active recombinant human erythropoietin expressed in hairy root cultures and regenerated plantlets of Nicotiana tabacum L.

Poornima Devi Gurusamy,Holger Schäfer,Michael Wink,Siva Ramamoorthy,Obul Reddy Bandapalli

doi:10.1371/journal.pone.0182367

Poornima Devi Gurusamy, Holger Schäfer + Show 3 more

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https://doi.org/10.1371/journal.pone.0182367

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Abstract

Hairy root culture is a potential alternative to conventional mammalian cell culture to produce recombinant proteins due to its ease in protein recovery, low costs and absence of potentially human pathogenic contaminants. The current study focussed to develop a new platform of a hairy root culture system from Nicotiana tabacum for the production of recombinant human EPO (rhEPO), which is regularly produced in mammalian cells. The human EPO construct was amplified with C-terminal hexahistidine tag from a cDNA of Caco-2 cells. Two versions of rhEPO clones, with or without the N-terminal calreticulin (cal) fusion sequence, were produced by cloning the amplified construct into gateway binary vector pK7WG2D. Following Agrobacterium rhizogenes mediated transformation of tobacco explants; integration and expression of constructs in hairy roots were confirmed by several tests at DNA, RNA and protein levels. The amount of intracellular rhEPO from hairy root cultures with cal signal peptide was measured up to 66.75 ng g-1 of total soluble protein. The presence of the ER signal peptide (cal) was essential for the secretion of rhEPO into the spent medium; no protein was detected from hairy root cultures without ER signal peptide. The addition of polyvinylpyrrolidone enhanced the stabilization of secreted rhEPO leading to a 5.6 fold increase to a maximum concentration of 185.48 pg rhEPOHR g-1 FW hairy root cultures. The rhizo-secreted rhEPO was separated by HPLC and its biological activity was confirmed by testing distinct parameters for proliferation and survival in retinal pigment epithelial cells (ARPE). In addition, the rhEPO was detected to an amount 14.8 ng g-1 of total soluble leaf protein in transgenic T0 generation plantlets regenerated from hairy root cultures with cal signal peptide.

Highlights

Erythropoietin (EPO) is a glycoprotein hormone, which influences the production of erythrocytes through a process called erythropoiesis [1]
In order to express biologically active rhEPO from the hairy root cultures (rhEPOHR) in N. tabacum, the EPO cDNA of 0.958 kb was amplified from Caucasian colon adenocarcinoma cells (Caco-2) cells using F1 and R1 primers (Fig 1A)
The amplified product was cloned in a pDrive (Qiagen) cloning vector, which was used as a template for sub-cloning in plant binary expression vector pK7WG2D

Summary

Introduction

Erythropoietin (EPO) is a glycoprotein hormone, which influences the production of erythrocytes through a process called erythropoiesis [1]. Branched hairy roots, which are covered with tiny root hairs, represent an interesting plant tissue culture system for the continuous production of recombinant proteins and secondary metabolites [25]. These cultures can grow on Murashige Skoog (MS) media [26] with sugars and do not require any hormones or vitamins for their growth [27]. We report an improved hairy root culture system of tobacco (Nicotiana tabacum) for the expression and secretion of biologically active rhEPO. Biological activity of the rhEPOHR secreted from hairy root cultures with cal signal peptide was determined using a retinal pigmented epithelial (ARPE) cells. The present study report a simple hairy root culture system for the in vitro production of biologically active rhEPOHR

Materials and methods

Results and discussion

Conclusion