Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana

Aditya Prakash Soni; Juhee Lee; Kunyoo Shin; Hisashi Koiwa; Inhwan Hwang

doi:10.3389/fpls.2022.922694

Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana

Front Plant Sci. 2022 May 31:13:922694. doi: 10.3389/fpls.2022.922694. eCollection 2022.

Authors

Aditya Prakash Soni¹, Juhee Lee^{1

2}, Kunyoo Shin^{1

2}, Hisashi Koiwa^{3

4}, Inhwan Hwang¹

Affiliations

¹ Department of Life Science, Pohang University of Science and Technology, Pohang, South Korea.
² Department of Biological Sciences, College of Natural Science, Seoul National University, Seoul, South Korea.
³ Molecular and Environmental Plant Sciences, Texas A&M University, College Station, TX, United States.
⁴ Vegetable and Fruit Development Center, Department of Horticultural Sciences, Texas A&M University, College Station, TX, United States.

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.

Keywords: CBM3; LAP-TGFβ1; Nicotiana benthamiana; acid activation; human TGFβ1; human growth factors; recombinant proteins.