Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.)

Iron is an essential element used by all eukaryotic organisms and most microorganisms as a cofactor for numerous proteins or enzymes involved in respiration, DNA synthesis, and many other critical metabolic processes [1]. Cellular iron deficiency can arrest cell proliferation and even cause cell death, whereas the excessive iron will be toxic to cells by reacting with oxygen via the Fenton reaction to produce highly reactive hydroxyl radicals that cause oxidative damage to cells [1, 2]. To overcome the dual challenges of iron deficiency and overload, a family of iron carrier glycoproteins collectively called transferrins has evolved in nearly all organisms to tightly control cellular iron uptake, storage, and transport to maintain cellular iron homeostasis [3]. The transferrin protein family includes serum transferrin (TF); lactoferrin (LF) found in mammalian extracellular secretions such as milk, tears, and pancreatic fluid; melanotransferrin (mTF) which is present on the surface of melanocytes and in liver and intestinal epithelium; and ovotransferrin (oTF) found in bird and reptile oviduct secretions and egg white. While all members of the transferrin protein family can bind iron to control free iron level, TF is currently the only protein that has been proven to be able to transport iron into cells [1]. TF is a single-chain glycoprotein of 679 amino acid residues including 38 cysteine residues which are all disulfide bonded. TF consists of two homologous halves, each comprising about 340 amino acid residues and sharing about 40% sequence identity [1, 4, 5]. The two homologous halves are shown by X-ray crystallography to fold into two distinct globular lobes called N- and C-terminal lobes [1, 4]. Each lobe comprises two dissimilar domains (N1 and N2 in the N- lobe; C1 and C2 in the C- lobe) separated by a deep cleft, where the iron binding site is located. The iron-binding ligands in each lobe are identical, which involves the side chains of an aspartic acid, two tyrosines, a histidine and two oxygen molecules from a synergistic carbonate anion [1, 2, 4–6]. The cellular iron uptake and transport is normally driven by a TF/TF receptor (TFR)-mediated endocytotic process [1]. When TF is free of iron (apo-TF), the two domains of each TF lobe (N1, N2 and C1, C2) remain apart from each other, forming a large water-filled cleft for easy access by the ferric iron. The apo-TF can then binds one (monoferric TF) or two iron molecules (diferric TF or holo-TF) by the coordination of iron-binding ligands at the extracellular pH of 7.4. The diferric TF then binds to TFR on the cell surface in a way that the TF C- lobe binds laterally to the helical domain of the dimeric TFR ectodomain while the TF N-lobe is sandwiched between the bulk of the dimeric TFR ectodomain and the cell membrane [7, 8]. This TF-TFR complex is then endocytosed into the early endosome, where the acidic environment (pH 5.5) triggers the conformational change of TF-TFR and the subsequent release of iron from TF by first protonating and dissociating the synergistic anion followed by protonating iron binding-related His and/or Tyr ligands [1, 6]. Finally, the apo-TF-TFR complex is recycled to the cell surface, where the neutral extracellular pH will dissociate the complex and release the TF for re-use. The TF-TFR complex-mediated endocytosis pathway of iron transport is not only biologically significant for maintaining cellular iron homeostasis, but also has important pharmaceutical applications. TF is a requisite component of almost all serum-free cell culture media to ensure iron delivery for propagating cells and maintaining sustained growth in mammalian culture for the production of therapeutic proteins and vaccines [9–12]. In addition, TF has also been actively pursued as a drug-delivery vehicle due to its unique receptor-mediated endocytosis pathway as well as its added advantages of being biodegradable, nontoxic, and nonimmunogenic [13–15]. TF not only can deliver anti-cancer drugs into primary proliferating malignant cells where the TFR is abundantly expressed [14], but also can deliver drugs to the brain by crossing the blood-brain barrier (BBB), which is a major barrier for administrated drugs to reach the central nervous system (CNS) [13,15, 16]. TF can also be exploited for the oral delivery of protein-based therapeutics [17, 18], as TF is resistant to proteolytic degradation and TFR is abundantly expressed in human gastrointestinal (GI) epithelium [17, 19]. With the increasing concerns over the risk of transmission of infectious pathogenic agents from the use of human or animal plasma-derived TFs in both cell culture and drug delivery applications, recombinant transferrin (rTF) is preferred to native TF [20]. Recombinant human TF (rhTF) has long been pursued in a variety of expression systems [21], but proves to be challenging largely due to hTF’s complicated structural characteristics as described above. The commonly used E. coli system for production of recombinant proteins has proved to be impractical for producing rhTF, as the expressed rhTF protein remains in insoluble inclusion bodies and the yield of functionally active rhTF after renaturation is very limited [22]. Although both the insect cell (baculovirus) [23] and mammalian cell [21] expression systems have been shown to be able to express the bioactive rhTF, neither of them can provide enough quantity to be commercially available because of the low expression level and the high cost of production. Recently, bioactive rhTF has been expressed in Saccharomyces Cerevisiae [24] and become commercially available. This yeast-derived rhTF, however, still remains very expensive (Millipore, Billerica, MA). To address the problems of both the shortage and the high cost of rhTF, alternative expression systems need to be explored. With the advancement of plant molecular biology in general and the improvement of plant transformation techniques in particular, plant hosts have become a powerful system to produce recombinant proteins cost-effectively and on a large scale [25–28]. In this paper, we report the high level expression of rhTF in rice grains, and the purification as well as the biochemical and functional characterization of rhTF. The expression level of rhTF is estimated at 1% seed dry weight. The rhTF was able to be extracted with saline buffer and purified by a one step anion exchange chromatographic process to greater than 95% purity. The rice-derived rhTF was shown to display similar structural characteristics and biological functionalities to that of native hTF.