Redox state of single chain Fv fragments targeted to the endoplasmic reticulum, cytosol and mitochondria.
In this paper we have engineered the targeting of ScFv fragments to mitochondria and demonstrated that this can occur efficiently. This extends the range of subcellular compartments where antibody domains can be targeted in order to interfere with the action of the corresponding antigen. Moreover, we have compared the redox state of ScFv fragments targeted to the secretory compartment, the cytosol and the mitochondria, and demonstrated that cysteine residues in ScFv targeted to the secretory compartments and to the mitochondria are oxidized. On the contrary, cytosolic antibody domains are expressed in a reduced state, which is probably the reason for their lower expression levels. These pitfalls, however, do not prevent their successful utilization for intracellular immunization.
Biocca
S
S
Institute of Neurobiology, CNR, Roma, Italy. silvia@biocell.irmkant.rm.cnr.it
Ruberti
F
F
Tafani
M
M
Pierandrei-Amaldi
P
P
Cattaneo
A
A
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000276:immunology*; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics; D008928:Mitochondria / Q000276:immunology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D021382:Protein Sorting Signals / Q000737:chemistry; D016283:Proto-Oncogene Proteins p21(ras) / Q000276:immunology; D011993:Recombinant Fusion Proteins; D014162:Transfection; D018631:ras Proteins
D004220:Disulfides; D007128:Immunoglobulin Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D003576:Electron Transport Complex IV; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D003545:Cysteine; D004229:Dithiothreitol