Production of recombinant active human TGFβ1 in Nicotiana benthamiana.

Abstract

The use of plant systems to produce recombinant proteins is gaining popularity. Plant-based recombinant protein manufacturing has a number of advantages, including low cost, high scalability, high product safety, and post-translational modification. Despite this, plant-based production systems must overcome numerous technical challenges in downstream processing as well as regulatory barriers in order to compete with traditional production platforms. Protein purification, on the other hand, is one of the most difficult procedures in the creation of recombinant proteins in plants. Various proteins derived from plants have been proved to be biologically active. Human growth factors and cytokines, on the other hand, are difficult to produce in heterologous systems because they frequently take complicated forms, such as homo- or heterodimers, and their synthesis is tightly regulated in vivo. Few of the growth factors such as TGFβs are very sensitive to their N-terminal partners to achieve correct folding and bioactivity. Out of various human growth factors, the production of recombinant human TGFβ1 has been extremely challenging and no plant-based purification system has been established so far. Although few attempts had been made in N. benthamiana, they either failed to purify or produced inclusion bodies which necessitated undergoing denaturation/refolding processes. In our present study, we successfully developed a method to produce a high level of soluble recombinant active TGFβ1 in N. benthamiana without undergoing denaturation/refolding in vitro, moreover, active dimers of TGFβ1 could be obtained by following simple steps. By addition of the M domain, recombinant TGFβ1 was expressed at a high level while CBM3 domain was used to selectively purify the recombinant protein. Out of various expression design cassettes, BMC:LAP[C33S]EK:TGFβ1 was found to be the best in terms of expression and solubility. After MCC-CBM3 mediated purification and on-bound cleavage by Enterokinase, TGFβ1 active dimers could be obtained by HCl treatment. The dimeric TGFβ1 could form perfect dimers and able to show biological activity and 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.