In order to achieve optimal biological activity and desired pharmacokinetic profiles, a dithiocyclopeptide linker was designed for in vivo release of protein domains from a recombinant fusion protein. This novel in vivo cleavable disulfide linker, based on a dithiocyclopeptide containing a thrombin-sensitive sequence and an intramolecular disulfide bond, was inserted between transferrin and granulocyte colony-stimulating factor (G-CSF) recombinant fusion protein domains. After expression of the fusion protein, G-C-T, from HEK293 cells, thrombin treatment in vitro generated a fusion protein linked via a reversible disulfide bond that was quickly cleaved in vivo, separating the protein domains. After release from the fusion protein, free G-CSF exhibited an improved biological activity in a cell proliferation assay. Although reversible disulfide bonds are commonly used in protein chemical conjugation methods, to our knowledge this report is the first example of the construction of a recombinant fusion protein with a disulfide linkage for the release of the functional domain. This linker design can be adapted to diverse recombinant fusion proteins in which in vivo separation of protein domains is required to achieve an improved therapeutic effect and a desirable pharmacokinetic profile and biodistribution of the functional domain.
Recombinant fusion proteins have become an important class of molecules in numerous fields of biotechnology, including protein engineering and purification, drug targeting and delivery, and immunology. By genetically fusing two or more genes together, the resulting fusion protein can attain multiple functional properties derived from each of its components (1). Successfully constructed fusion proteins require not only the desired component proteins, but also suitable linkers to connect the protein domains. Linkers in fusion proteins generally consist of stable peptide sequences, including the glycine-serine linker (GGGGS)n and α-helix–forming peptide linkers, such as A(EAAAK)nA (n = 2–5), which can provide structure flexibility, improve protein stability, or increase biological activity (2,3,4,5). However, stable peptide linkers do not allow for the separation of the two fusion protein domains in vivo and have several limitations including steric hindrance between two functional domains, altered biodistribution and metabolism of the protein moieties due to interference with each other, and incorrect folding of the fusion protein (6).
To overcome some of these potential pitfalls in stable peptide linkers, we designed a fusion protein linkage utilizing the reversible nature of the disulfide bond (7). There are numerous examples of application of a disulfide linkage between two proteins in drug delivery, either by using cysteine amino acid residues in proteins or by employing hetero- or homobifunctional linkers (8,9,10). This approach requires chemical conjugation of the two separate proteins, since recombinant technologies require expression of fusion proteins as one single polypeptide chain. However, the major obstacle for the chemical conjugation approach is that the composition and size of the final product can be heterogeneous, which is unacceptable for therapeutic use. In comparison, the novel recombinant fusion protein approach described in this report offers the significant advantage in the generation of a precisely constructed, homogeneous product. This cleavable dithiocyclopeptide linker contains a thrombin-sensitive sequence, as well as an intramolecular disulfide bond formed between two cysteine residues of the linker. Treatment in vitro with thrombin results in cleavage of the thrombin-sensitive sequence, while the disulfide linkage between the two domains of the recombinant fusion protein remains (Figure 1A). Conceivably, the disulfide linkage will be reduced in vivo, allowing for the protein domains to be separated and regain their individual characteristics such as biological activity, biodistribution, and/or metabolism. In this paper, we use a fusion protein containing granulocyte colony-stimulating factor (G-CSF) and transferrin (Tf) (11) to demonstrate the successful construction and the in vivo cleavability of a dithiocyclopeptide linker between two domains of a recombinant fusion protein.
Materials and methods Cell lines
HEK293 cells, purchased from ATCC (Manassas, VA, USA), were grown as monolayers in DMEM with 10% (v/v) FBS at 37°C in 5% CO2. Protein-free, chemically defined CD293 medium, obtained from Invitrogen (Carlsbad, CA, USA), was used for protein expression after transfection of HEK293 cells. Murine myeloblastic NFS60 cells, provided by James Ihle (St. Jude Children's Research Hospital, Memphis, TN, USA), were grown in RPMI medium 1640 (Sigma-Aldrich, St. Louis, MO, USA) with 10% (v/v) FBS and 0.1 ng/mL recombinant mouse interleukin-3 (IL-3).Animals
Male CF-1 mice (18–20 g) from Charles River Laboratories (Kingston, NY, USA) were used for in vivo cleavability studies. The protocol of animal experiments in this study has been approved by the Institutional Animal Care and Use Committee (IACUC) at USC. The animals were handled in accordance with the “Guide for the Care and Use of Laboratory Animals” [National Institutes of Health (NIH) Publication no.85–23, revised 1985]. The animals were fed a standard laboratory rodent diet (Purina Mills, St. Louis, MO, USA) and housed on a 12-h light/dark cycle with room temperature maintained at 22° ± 3°C and relative humidity at 50% ± 20%.Construction of G-CSF-dithiocyclopeptide-Tf (G-C-T) plasmid
G-CSF-Tffusion protein was constructed into the mammalian expression vector pcDNA3.0 (Invitrogen) as previously described (11). A dipeptide linker, Leu-Glu, was introduced between G-CSF and Tf due to the cloning site XhoI. The design of dithiocyclopeptide linker was based on the structure of the cyclopeptide, somatostatin, with the replacement of amino acids 8–10, WKT, by a thrombin-specific sequence, PRS. Two cysteine residues on somatostatin, Cys-3 and Cys-14, naturally form a disulfide bond. The oligonucleotides encoding the dithiocyclopeptide linker, synthesized by Invitrogen, were inserted between G-CSF and Tf by using the XhoI cloning site. The correct sequence was confirmed by DNA sequencing.