Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana
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 ofte...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-05-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2022.922694/full |
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| author | Aditya Prakash Soni Juhee Lee Juhee Lee Kunyoo Shin Kunyoo Shin Hisashi Koiwa Hisashi Koiwa Inhwan Hwang |
| author_facet | Aditya Prakash Soni Juhee Lee Juhee Lee Kunyoo Shin Kunyoo Shin Hisashi Koiwa Hisashi Koiwa Inhwan Hwang |
| author_sort | Aditya Prakash Soni |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T22:06:42Z |
| format | Article |
| id | doaj.art-e0b871a47c27495e8dca98977083dd23 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-13T22:06:42Z |
| publishDate | 2022-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-e0b871a47c27495e8dca98977083dd232022-12-22T02:27:56ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2022-05-011310.3389/fpls.2022.922694922694Production of Recombinant Active Human TGFβ1 in Nicotiana benthamianaAditya Prakash Soni0Juhee Lee1Juhee Lee2Kunyoo Shin3Kunyoo Shin4Hisashi Koiwa5Hisashi Koiwa6Inhwan Hwang7Department of Life Science, Pohang University of Science and Technology, Pohang, South KoreaDepartment of Life Science, Pohang University of Science and Technology, Pohang, South KoreaDepartment of Biological Sciences, College of Natural Science, Seoul National University, Seoul, South KoreaDepartment of Life Science, Pohang University of Science and Technology, Pohang, South KoreaDepartment of Biological Sciences, College of Natural Science, Seoul National University, Seoul, South KoreaMolecular and Environmental Plant Sciences, Texas A&M University, College Station, TX, United StatesVegetable and Fruit Development Center, Department of Horticultural Sciences, Texas A&M University, College Station, TX, United StatesDepartment of Life Science, Pohang University of Science and Technology, Pohang, South KoreaThe 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.https://www.frontiersin.org/articles/10.3389/fpls.2022.922694/fullrecombinant proteinshuman growth factorsLAP-TGFβ1human TGFβ1Nicotiana benthamianaCBM3 |
| spellingShingle | Aditya Prakash Soni Juhee Lee Juhee Lee Kunyoo Shin Kunyoo Shin Hisashi Koiwa Hisashi Koiwa Inhwan Hwang Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana Frontiers in Plant Science recombinant proteins human growth factors LAP-TGFβ1 human TGFβ1 Nicotiana benthamiana CBM3 |
| title | Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana |
| title_full | Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana |
| title_fullStr | Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana |
| title_full_unstemmed | Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana |
| title_short | Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana |
| title_sort | production of recombinant active human tgfβ1 in nicotiana benthamiana |
| topic | recombinant proteins human growth factors LAP-TGFβ1 human TGFβ1 Nicotiana benthamiana CBM3 |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2022.922694/full |
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