Plant-derived recombinant human serum transferrin demonstrates multiple functions Martin E. Brandsma 1 , Hong Diao 2 , Xiaofeng Wang 1 , Susanne E. Kohalmi 1 , Anthony M. Jevnikar 2 and Shengwu Ma 1,2,3, * 1 Department of Biology, University of Western Ontario, London, Ontario, Canada 2 Transplantation Immunology Group, Lawson Health Research Institute, London, Ontario, Canada 3 Plantigen Inc., 346 South Street, London, Ontario, Canada Received 14 August 2009; revised 16 November 2009; accepted 28 November 2009. *Correspondence (Tel +1(519)685 8500 ext. 75855; fax +1(519)432 7367; email sma@uwo.ca) Keywords: Plant bioreactor, human serum transferrin, multitasking pro- tein, drug carrier, fusion protein. Summary Human serum transferrin (hTf) is the major iron-binding protein in human plasma, having a vital role in iron transport. Additionally, hTf has many other uses including antimicrobial functions and growth factor effects on mammalian cell proliferation and differentiation. The multitask nature of hTf makes it highly valuable for different therapeutic and commercial applications. However, the success of hTf in these appli- cations is critically dependent on the availability of high-quality hTf in large amounts. In this study, we have developed plants as a novel platform for the production of recombinant (r)hTf. We show here that transgenic plants are an efficient system for rhTf production, with a maximum accumulation of 0.25% total soluble protein (TSP) (or up to 33.5 lg ⁄ g fresh leaf weight). Furthermore, plant-derived rhTf retains many of the biological activities synonymous with native hTf. In particular, rhTf reversibly binds iron in vitro, exhibits bacteriostatic activity, supports cell proliferation in serum- free medium and can be internalized into mammalian cells in vitro. The success of this study validates the future application of plant rhTf in a variety of fields. Of par- ticular interest is the use of plant rhTf as a novel carrier for cell-specific or oral deliv- ery of protein ⁄ peptide drugs for the treatment of human diseases such as diabetes. To demonstrate this hypothesis, we have additionally expressed an hTf fusion protein containing glucagon-like peptide 1 (GLP-1) or its derivative in plants. Here, we show that plant-derived hTf-GLP-1 fusion proteins retain the ability to be internalized by mammalian cells when added to culture medium in vitro. Introduction Transferrins (Tf) comprise a family of homologous iron- binding glycoproteins found in all vertebrates (Aisen and Harris, 1989), whose primary function is iron sequestration and transport. Tf is a monomeric protein with a molecular mass ranging from 76 to 81 kDa depending on the extent of glycosylation. Each Tf protein consists of two similar lobes named the N- and C-terminal respectively, with each lobe containing a single iron-binding site (Aisen and Harris, 1989; Baker et al., 2002). Human serum transferrin (hTf) is a major member of the Tf protein family. The hTf protein is composed of 679 amino acids and is synthesized primarily in the liver to be secreted into the blood (MacGil- livray et al., 1983). The primary function of hTf is to sequester free iron within the blood and to subsequently transport it throughout the body (MacGillivray et al., 1983). Radioactive tracer studies indicate that at least 80% of the iron bound to hTf is delivered to the bone marrow and incorporated into newly formed erythrocytes (Finch and Huebers, 1982). In addition to its well-known iron transport function, hTf has numerous additional func- tions, many of them not related to its iron-carrying capac- ity. For example, hTf has been shown to promote the ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd 489 Plant Biotechnology Journal (2010) 8, pp. 489–505 doi: 10.1111/j.1467-7652.2010.00499.x