Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.)

Abstract

The Fe3+ binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin™ (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin™) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin™ is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin™ is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin™ bind Fe3+ tightly yet reversibly. Although previously shown to be capable of delivering Fe3+ to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin™ might be suitable for consideration in biopharmaceutical applications.

Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativaL.)

“…This result is consistent with our previous finding in a competitive immunoassay that rhTF is equivalent to baby hamster kidney (BHK) cell derived recombinant N-His hTF in its ability to bind to the soluble portion of the TFR [15]. It is also in good agreement with other reports that hTF’s internalization increases linearly with time in enterocyte-like Caco-2 cells due to this type of cells’ unique accumulation of TF [29].…”

“…Our previous cell-free biochemical and biophysical studies show that both lobes of rhTF bind Fe 3+ tightly yet reversibly similarly to hTF [15,25]. In this study we further characterize the TFR binding, TFR-mediated endocytosis and iron delivery function of rhTF using cell-based assays.…”

“…β-phase) of rice-derived rhTF, yeast-derived aglycosylated rhTF and hTF are 14.8, 13.8, and 18.6 hr, respectively (Unpublished data). The relatively shorter serum half-life could be due to the lack of N-linked glycans in the two recombinant transferrins [14,15,25]. Nevertheless, rice-derived rhTF is shown to have a sufficiently long serum half-life compared to native hTF.…”

“…With the increasing concerns over the risk of transmission of infectious pathogenic agents from the use of human and animal plasma-derived TFs in both cell culture and drug delivery applications [13-15], rhTF has long been pursued in a variety of expression systems [16-24]. However, expression of fully functional TF has been proven to be challenging largely due to hTF’s complicated structural characteristics including 19 disulfide bonds and two homologous lobes (N-lobe and C-lobe).…”

“…Expression yield is 40% of total soluble protein or 1% seed dry weight (10 g/kg) [25]. The rice-derived rhTF is shown to be biochemically and structurally similar to hTF and mammalian cell-derived rhTF, and able to bind Fe 3+ tightly yet reversibly [15]. In the present work, we have characterized rice-derived rhTF’s TFR-binding, TFR-mediated endocytosis and intracellular processing, and its cellular iron delivery to diverse mammalian cells through various cell-based assays.…”