A transferrin-based fusion protein, proinsulin-transferrin (ProIns-Tf) had been constructed using recombinant protein technology in our laboratory. Based on the in vitro preliminary results, ProIns-Tf reduced glucose production in H4IIE liver cells and increased glucose uptake in adipocytes. These potent effects were achieved by conversion of the ProIns moiety to an active Ins-like moiety through the mechanism of TfR-mediated endocytosis. Therefore, ProIns-Tf showed great potential for diabetes therapeutics. However, it was also observed that the preparation of this fusion protein also produced a large amount of protein impurities which might have impeding effects on the further characterization studies.

In this report, ProIns-Tf fusion protein was purified using the polyhistidine tag technique. The hexahistidine tag (6xHis tag) was bound to the C-terminus of Tf domain through mutagenic polymerase chain reaction (PCR) and recombinant DNA technology. After the protein expression and purification processes, the protein analysis results indicated that the purity of ProIns-Tf was significantly enhanced. In addition, through the transferrin receptor (TfR) competitive binding assay, we found that the insertion of C-terminal 6xHis tag did not interfere with the binding affinity of Tf domain to TfR. Therefore, it was concluded that the His-tag purification is a suitable approach to improve the purity of Tf-based fusion protein.

Furthermore, by comparing the results to previous studies in our laboratory, we also noticed that the TfR binding affinity of our Tf-based fusion proteins – ProIns-Tf, granulocyte colony-stimulating factor- Tf (G-CSF-Tf) and human growth hormone-Tf (hGH-Tf) – had a positive correlation to the molecular weight of the bound protein domain. It also implies that the beneficial effects of the TfR-mediated endocytosis should be limited by the size of Tf-bound protein drug. As a result, molecular weight of protein drug will be considered as an important criterion for the design of future Tf-based recombinant fusion proteins.