Engineering Refolded Transferrin for Iron Sequestration: Expression, Purification, and Functional Characterization of Recombinant Human Transferrin
Siah Shin
Seoul International School, Seoul, South Korea
Publication date: July 10, 2026
Seoul International School, Seoul, South Korea
Publication date: July 10, 2026
DOI: http://doi.org/10.34614/JIYRC2026I03
ABSTRACT
Transferrin is the primary iron-transport glycoprotein in human plasma, composed of two homologous lobes that independently bind ferric ions and regulate systemic iron homeostasis. Despite its biological significance and growing therapeutic relevance as a drug-delivery scaffold, recombinant production of human transferrin remains challenging due to consistent accumulation in inclusion bodies during Escherichia coli expression. In this study, we established a unified workflow to produce and compare three transferrin variants—the N-terminal (20–355 a.a.), C-terminal (356–699 a.a.), and full-length protein (20–699 a.a.)—under identical expression, purification, and refolding conditions. All constructs were cloned from human cancer cell line cDNA, expressed in E. coli BL21(DE3), purified from inclusion bodies under denaturing conditions, and renatured using a controlled step-dialysis process. SDS–PAGE analysis confirmed high purity and minimal aggregation following refolding, eliminating the need for affinity chromatography. Functional assays revealed that the three variants exhibit distinct iron-protective properties: full-length transferrin showed the strongest rescue of RGC5 neuronal cells exposed to high iron concentrations, whereas the individual N- and C-terminal Tf proteins displayed partial but measurable protection. These results provide a validated biochemical platform for transferrin reconstruction and highlight the importance of intact domain cooperativity for optimal iron sequestration and cellular protection.
Transferrin is the primary iron-transport glycoprotein in human plasma, composed of two homologous lobes that independently bind ferric ions and regulate systemic iron homeostasis. Despite its biological significance and growing therapeutic relevance as a drug-delivery scaffold, recombinant production of human transferrin remains challenging due to consistent accumulation in inclusion bodies during Escherichia coli expression. In this study, we established a unified workflow to produce and compare three transferrin variants—the N-terminal (20–355 a.a.), C-terminal (356–699 a.a.), and full-length protein (20–699 a.a.)—under identical expression, purification, and refolding conditions. All constructs were cloned from human cancer cell line cDNA, expressed in E. coli BL21(DE3), purified from inclusion bodies under denaturing conditions, and renatured using a controlled step-dialysis process. SDS–PAGE analysis confirmed high purity and minimal aggregation following refolding, eliminating the need for affinity chromatography. Functional assays revealed that the three variants exhibit distinct iron-protective properties: full-length transferrin showed the strongest rescue of RGC5 neuronal cells exposed to high iron concentrations, whereas the individual N- and C-terminal Tf proteins displayed partial but measurable protection. These results provide a validated biochemical platform for transferrin reconstruction and highlight the importance of intact domain cooperativity for optimal iron sequestration and cellular protection.
