Purpose To demonstrate a high-level expression of transferrin (Tf)-based fusion proteins by inserting a helical linker between two protein domains. Methods Tf-based fusion proteins were designed to contain oligonucleotides encoding a helical linker inserted between the protein domains. Plasmid constructs were transfected into HEK293 cells and the secreted fusion proteins were purified from conditioned serum free media. Expression was assessed using both SDS-PAGE and Western Blot using anti-hGH, G-CSF, or Tf antibodies; protein bands were analyzed using Quantity One software. The function of fusion proteins consisting of human growth hormone (hGH) and Tf was evaluated in Nb2 cell proliferation assays. Results The fusion proteins containing a helical linker, hGH-(H4)2-Tf and Tf-(H4)2-hGH, were expressed 1.7- and 2.4-fold higher, respectively, with a 2-fold lower ED50 than the hGH-Tf fusion protein without a helical linker. The Tf-(H4)2-G-CSF fusion protein exhibited a greater expression with an 11.2-fold increase compared with Tf-G-CSF fusion protein. Conclusions The helical linker introduced in Tf-fusion proteins resulted in a high-level of expression with improved in vitro bioactivity. This approach provides a simple method to increase poor expression of other fusion proteins.

BackgroundTransferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications.ResultThrough a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production.ConclusionThe functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.

In contrast to the wide applications of recombinant bifunctional fusion proteins in clinical usage, the systematic study for the pharmacokinetics (PK) of bifunctional fusion proteins is left blank. In this report, recombinant fusion proteins consisting of transferrin (Tf) and growth hormone (GH) or granulocyte colony-stimulating factor (G-CSF) have been constructed as a model for studying the PK of bifunctional fusion proteins. The results showed that the insertion of different linkers between the two protein domains altered the binding affinities of the fusion proteins to both domain receptors, and that the fusion proteins’ plasma half-lives were greatly affected. A strong correlation between GH receptor binding affinity and plasma half-life of GH-Tf fusion proteins was observed. In addition, we demonstrated that the intracellular processing after receptor binding plays an important role in determining the half-life of fusion proteins. While the binding of the GH domain to the GH receptor will lead to endocytosis and lysosomal degradation in target cells, binding of the Tf domain to the Tf receptor may recycle the fusion protein and prolong its plasma half-life. To further confirm the effects of receptor binding on plasma half-life, G-CSF-Tf bifunctional fusion proteins with the same three linkers as GH-Tf were evaluated. While the 3 fusion proteins showed a similar G-CSF receptor binding affinity, the G-CSF-Tf fusion protein with the higher Tf receptor binding affinity exhibited longer plasma half-life. The linker insertion further demonstrated the involvement of Tf in recycling and prolonging plasma half-life. Based on our results, a model was developed to summarize the factors in determining the PK of the bifunctional fusion proteins. Our findings are useful for predicting the plasma half-lives, as well as for improving the pharmacokinetic profiles of therapeutic bifunctional fusion proteins by applying linker technology.