Cytoplasmic expression and specific binding of the VH/VL single domain intrabodies in transfected NIH3T3 cells.
Intracellularly expressed antibody fragments (intrabodies) have been utilized as powerful tools not only for clinical applications but also for the functional analysis of proteins inside the cell. Among several types of intrabodies developed so far, single domain types composed of only the variable regions (V(H) or V(L)) of antibodies are the smallest and thus the easiest to design. In this study, four types of single domain intrabodies were evaluated against a cytosolic protein, Wiskott-Aldrich syndrome protein (WASP), in gene-transfected NIH3T3 cells. These single domains were composed of the V(H) and V(L) region with or without their leader sequences. Although these single domains were expressed at similar levels in NIH3T3 cells, the binding activity to the cytosolic target was higher in the single domain constructs with leader sequences. These results suggest the usefulness of the single domain intrabody constructs to analyze the functional domains of cytosolic proteins in cells.
Sawahata
Ryoko
R
Transgenic Animal Research Center, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
Sato
Mitsuru
M
Kitani
Hiroshi
H
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D014162:Transfection; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D051316:Wiskott-Aldrich Syndrome Protein