Fusion Proteins With Cleavable Spacers and Uses Thereof
a technology of fusion proteins and spacers, applied in the field of fusion proteins, can solve the problems of poor compliance, adverse side effects of patients, and difficulty in achieving oral delivery of these therapeutic agents into the tissues of choice or across epithelial barriers of choi
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example i
[0069]Using receptors as targets and receptor-binding ligands as vectors for transcellular transport is a promising way of achieving selective delivery of peptide and protein drugs across the intestinal epithelium [7]. This process, termed receptor-mediated transcytosis, is highly specific because it enhances only the transport of molecules that are conjugated to receptor-binding ligands [8]. Receptor-mediated transcytosis is an inherent cellular process in epithelial and endothelial cells [9]. Unlike most current approaches, which use penetration enhancers such as bile salts and lipids to increase epithelial insulin absorption [10], a receptor-mediated transcytotic process does not change the structure of the plasma membranes or the intercellular junctions, and conceivably has fewer unwanted side-effects and safety concerns.
[0070]TfR has been utilized for the development of orally administered, receptor-mediated delivery systems for peptide and protein drugs for the following reaso...
example ii
[0163]In vitro Characterization of Fusion Protein with a Cyclodisulfide Peptide Linker
[0164]The fusion protein was produced by transiently transfecting HEK293 cells with plasmids encoding G-CSF-cyclodisuldiepeptide-transferrin (G-C-T). The dithiocyclopeptide linker in the fusion protein contained a thrombin-cutting sequence, PRS, and was characterized by the treatment with or without thrombin and / or dithiothreitol (DTT) followed by anti-G-CSF Western blotting analysis. For the thrombin treatment, 1 μg fusion protein was cleaved by 0.25 NIH unit thrombin when incubated at 20° C. for 16 h. For the DTT treatment, the thrombin-treated or intact fusion protein was added into the reducing loading buffer and boiled for 10 min to reduce the disulfide bond. Protein samples with or without thrombin and / or DTT treatment were then loaded into non-reducing SDS-PAGE and analyzed by anti-G-CSF Western blotting.
[0165]The Western blotting result showed that the disulfide bond formed between the two ...
example iii
Release of Free G-CSF from G-CSF-CYCLO-TF Fusion Protein Upon Treatment with Trypsin and Dithiothreitol
[0168]Method: 0.3 μg G-CSF-cyclo-Tf fusion protein was incubated with different amount of trypsin at 37° C. for 5 min. The fusion protein was then treated with DTT, loaded to reducing SDS-PAGE, and analyzed by anti-G-CSF Western blot.
[0169]Result: Under the suitable trypsin concentration (3 unit / ml, or 10 unit / ml), the cyclic linker was cleaved by trypsin as demonstrated by the appearance of the free G-CSF after DTT reduction (FIG. 14).
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