Design of an in vivo cleavable disulfide linker in recombinant fusion proteins.
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.
Chen
Xiaoying
X
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California School of Pharmacy, Los Angeles, CA 90089, USA.
Bai
Yun
Y
Zaro
Jennica L
JL
Shen
Wei-Chiang
WC
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D004220:Disulfides / Q000378:metabolism*; D015870:Gene Expression; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D010456:Peptides, Cyclic / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins; D013917:Thrombin / Q000378:metabolism; D014168:Transferrin / Q000235:genetics* / Q000378:metabolism
D004220:Disulfides; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014168:Transferrin; D016179:Granulocyte Colony-Stimulating Factor; D013917:Thrombin