Soni Aditya Prakash, Lee Juhee, Shin Kunyoo, Koiwa Hisashi, Hwang Inhwan
Department of Life Science, Pohang University of Science and Technology, Pohang, South Korea.
Department of Biological Sciences, College of Natural Science, Seoul National University, Seoul, South Korea.
Front Plant Sci. 2022 May 31;13:922694. doi: 10.3389/fpls.2022.922694. eCollection 2022.
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 this study, we demonstrated that the mature form of human TGFβ1 produced and purified from 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 . . Of these, a recombinant construct, β, was expressed at a high level in . . 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 and , 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 a few steps.
在植物系统中生产重组蛋白正受到越来越广泛的关注。事实上，各种植物生产的药物已被证明具有生物活性。然而，在异源系统中生产人类生长因子和细胞因子仍然具有挑战性，因为它们通常以复杂的形式存在，如同源或异源二聚体，并且它们的生产受到严格调控。在本研究中，我们证明了从植物中生产和纯化的人TGFβ1成熟形式在动物细胞中具有生物活性。为了生产TGFβ1的成熟形式，构建了各种包含TGFβ1成熟形式的重组基因并在植物中进行表达。其中，一个重组构建体β在植物中高水平表达。重组蛋白使用纤维素结合模块3（CBM3）作为亲和标签和微晶纤维素（MCC）珠作为基质进行一步纯化。结合在MCC珠上的TGFβ1重组蛋白用肠激酶进行蛋白水解处理以分离成熟的TGFβ1。通过盐酸处理释放仍与固定在MCC珠上的潜伏相关蛋白[LAP（C33S）]结合的成熟TGFβ1。纯化的TGFβ1激活了A549细胞系中TGFβ1介导的信号传导，从而诱导SMAD - 2的磷酸化、某些基因的表达以及丝状伪足样结构的形成。基于这些结果，我们提出，活性成熟TGFβ1作为在异源系统中生产最具挑战性的生长因子之一，可以通过几个步骤从植物中以高纯度生产出来。
