title: Conjugation to octa-arginine via disulfide bonds confers solubility to denatured proteins in physiological solution and enables efficient cell internalization.
abstract: Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa-arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate-buffered saline. However, almost all the R8-conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary-K1, Cos-7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8-conjugated β-galactosidase and R8-conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen-presenting cells and will benefit research and innovation in vaccine design and discovery.
authors.affiliation: Medinet Medical Institute, MEDINET, Setagaya-ku, Tokyo, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Kuwada
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Tadaki
authors.forename: Kaori
authors.initials: K
authors.lastname: Kambara
authors.forename: Kohji
authors.initials: K
authors.lastname: Egawa
authors.forename: Katsuo
authors.initials: K
authors.lastname: Noguchi
mesh_terms: D000818:Animals; D019556:COS Cells / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003713:Dendritic Cells / Q000378:metabolism; D003956:Dialysis; D004220:Disulfides / Q000737:chemistry*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000737:chemistry; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D010047:Ovalbumin / Q000737:chemistry; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D014508:Urea / Q000737:chemistry; D001616:beta-Galactosidase / Q000737:chemistry
chemical_list: D004220:Disulfides; D009842:Oligopeptides; D011994:Recombinant Proteins; C500307:octa-arginine peptide; D014508:Urea; D010047:Ovalbumin; D008156:Luciferases; D001616:beta-Galactosidase