Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana

Abstract

The production of recombinant proteins in plant systems is receiving wider attention. Indeed, various plant-produced pharmaceuticals have been shown to be biologically active. However, the production of human growth factors and cytokines in heterologous systems is still challenging because they often act as complex forms, such as homo- or hetero-dimers, and their production is tightly regulated in vivo. In this study, we demonstrated that the mature form of human TGFβ1 produced and purified from Nicotiana benthamiana shows biological activity in animal cells. To produce the mature form of TGFβ1, various recombinant genes containing the mature form of TGFβ1 were generated and produced in N. benthamiana. Of these, a recombinant construct, BiP:M:CBM3:LAP[C33S]:EK:TGFβ1, was expressed at a high level in N. benthamiana. Recombinant proteins were one-step purified using cellulose-binding module 3 (CBM3) as an affinity tag and microcrystalline cellulose (MCC) beads as a matrix. The TGFβ1 recombinant protein bound on MCC beads was proteolytically processed with enterokinase to separate mature TGFβ1. The mature TGFβ1 still associated with Latency Associated Protein, [LAP(C33S)] that had been immobilized on MCC beads was released by HCl treatment. Purified TGFβ1 activated TGFβ1-mediated signaling in the A549 cell line, thereby inducing phosphorylation of SMAD-2, the expression of ZEB-2 and SNAIL1, and the formation of a filopodia-like structure. Based on these results, we propose that active mature TGFβ1, one of the most challenging growth factors to produce in heterologous systems, can be produced from plants at a high degree of purity via a few steps.

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture

“…The complex disulfide structure and the involvement of the propeptide in native activin formation pose a significant challenge for refolding, making this protein an intriguing candidate for testing the reverse screening approach [23]. Previous studies have demonstrated the production of human TGFβ1 in Nicotiana benthamiana, the mature form with biological activity obtained after enterokinase treatment [38]. Based on the specific cleavage property of enterokinase, the fusion protein is released after treatment with this enzyme [39,40].…”

Nicotiana benthamiana as a potential source for producing anti-dengue virus D54 neutralizing therapeutic antibody

“…Plant expression systems offer a promising solution for anti-DENV mAb production. Among these, N. benthamiana stands out as one of the most well-characterized plants for recombinant proteins synthesis, like growth factors, enzymes, antibodies, and vaccines [ 28 , 29 ]. It provides unique benefits such as low operating costs, a rapid and simple production process, high reproducibility, ease of scalability, and the ability to express complex protein [ 30 , 31 ].…”

Artificial intelligence-driven systems engineering for next-generation plant-derived biopharmaceuticals

“…for easy purification and analysis. Immobilized metal-ion affinity chromatography is widely used for purification of hexahistidine tagged proteins ( Vafaee and Alizadeh, 2018 ; Islam et al., 2019 ; Hanittinan et al., 2020 ; Islam et al., 2020 ; Marques et al., 2020 ; Soni et al., 2022 ). Other techniques such as one-step cation-exchange chromatography, Protein G-/A-based affinity chromatography, diafiltration (antibody purification) and polyelectrolyte precipitation (removal of plant proteins), hydrophobic interaction chromatography (HIC) followed by hydrophobic charge induction chromatography (HCIC) are employed in recombinant plant protein purification ( Fulton et al., 2015 ; Park et al., 2015 ; Shi et al., 2019 ; Miura et al., 2020 ; Lim et al., 2022 ; Grandits et al., 2023 ).…”