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

Abstract

Transferrin is an essential ingredient used in cell culture media due to its crucial role in regulating cellular iron uptake, transport, and utilization. It is also a promising drug carrier used to increase a drug’s therapeutic index via the unique transferrin receptor-mediated endocytosis pathway. Due to the high risk of contamination with blood-borne pathogens from the use of human- or animal plasma-derived transferrin, recombinant transferrin is preferred for use as a replacement for native transferrin. We expressed recombinant human transferrin in rice (Oryza sativa L.) at a high level of 1% seed dry weight (10 g/kg). The recombinant human transferrin was able to be extracted with saline buffers and then purified by a one step anion exchange chromatographic process to greater than 95% purity. The rice-derived recombinant human transferrin was shown to be not only structurally similar to the native human transferrin, but also functionally the same as native transferrin in terms of reversible iron binding and promoting cell growth and productivity. These results indicate that rice-derived recombinant human transferrin should be a safe and low cost alternative to human or animal plasma-derived transferrin for use in cell culture-based biopharmaceutical production of protein therapeutics and vaccines.

Purification of transferrin by magnetic immunoaffinity beads

“…They reached 95% purity with a single step anion exchange chromatography and 90% purity with hydrophobic interaction chromatography [1]. For the immunoaffinity purification of serum Tf, Barroso et al have prepared anti-Tf antibody immobilized using silica-hydrazide columns and CNBr activatedsepharose columns.…”

Strategies to Increase Heterologous Protein Expression in Rice Grains

“…The protein composition of the endosperm is 60-70 % glutelins, 25-30 % prolamins, 5-10 % globulins (Glbs), and 0-5 % other proteins, depending on the rice variety. Protein yields have received considerable attention as they play a key role in the performance of the production platform (Zhang et al 2010;Broz et al 2013). The most common strategies to achieve a higher yield of the recombinant protein in rice seeds are by optimizing the promoter region, the untranslated regions, translation efficiency, subcellular localization/targeting of the target protein, codon optimization, knockdown or antisense technology, stable integration of the gene and its copy number, screening, selection, and breeding.…”

“…Many rice seed-specific promoters have been characterized for their activity, and among them, the glutelin, Glb, and 10-and 16-kDa prolamin promoters have shown to give the highest expression levels ranging from 6 to 15 % of total seed protein (1-2 % total seed weight; Nandi et al 2002;Huang et al 2002;Qu and Takaiwa, 2004;Wakasa et al 2006a;Wu et al 1998;Zhang et al 2010). The use of strong spatiotemporal promoters helps expression of protein at specific times during seed development.…”

“…This has been confirmed by several laboratories (Akashi 2001;Davis 1999;Rouwendel et al 1997). In the expression of human lysozyme, lactoferrin, and transferrin in rice grain, the codons were optimized for all three synthetic genes (Nandi and Huang 2009;Zhang et al 2010). For example, in the expression of recombinant human lactoferrin in rice, a total of 92 out of 130 codons were modified resulting in the G+C content being raised from 46 to 68 %.…”

Recovery of Recombinant Proteins from Plants Using Aqueous Two-Phase Partitioning Systems: An Outline

“…Corn and rice are popular seed crops for producing recombinant proteins. Corn has been used in commercial production for such recombinant proteins as avidin [14], aprotinin [15,16], β-GUS [17], and dog gastric lipase [18], while rice has been used to express human lysozyme [19], transferrin [20], and lactoferrin [21]. For leaf expression system, tobacco is the most popular host for generating recombinant proteins, such as lysozyme [22], β-glucuronidase [23][24][25], and monoclonal antibodies [26].…”