A universal strategy for stable intracellular antibodies.
The expression of intracellular antibodies (intrabodies) in mammalian cells has provided a powerful tool to manipulate microbial and cellular signalling pathways in a highly precise manner. However, several technical hurdles have thus far restricted their more widespread use. In particular, single-chain antibodies (scFvs) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use scFv-phage libraries as a source of intrabodies unless a preselection step was applied to identify these rare scFvs that could fold properly in the absence of disulfide bonds. Recently, we reported that scFvs can be efficiently expressed within the cytoplasm of bacteria when fused at the C-terminus of the Escherichia coli maltose-binding protein (MBP). Here, we demonstrate that such MBP-scFvs are similarly stabilized when expressed in the mammalian cell cytoplasm as well as other compartments. This was demonstrated by comparing MBP-scFv fusions to the corresponding unfused scFvs that activate a defective beta-galactosidase enzyme, others that neutralize the wild-type beta-galactosidase enzyme, and an antibody that blocks the epidermal growth factor receptor. In all cases, the MBP-scFvs significantly outperformed their unfused counterparts. Our results suggest that fusion of scFvs to MBP, and possibly to other "chaperones in the context of a fusion protein", may provide a universal approach for efficient expression of intrabodies in the mammalian cell cytoplasm. This strategy should allow investigators to bypass much of the in vitro scFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic scFv-phage libraries already in existence to identify intrabodies that will be active in vivo.
Shaki-Loewenstein
Shelly
S
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Green Building, Room 202, Tel-Aviv University, Ramat Aviv 69978, Israel.
Zfania
Rahely
R
Hyland
Stephen
S
Wels
Winfried S
WS
Benhar
Itai
I
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D066246:ErbB Receptors / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007424:Intracellular Fluid / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D007763:Lac Operon; D017510:Protein Folding; D001616:beta-Galactosidase / Q000378:metabolism
D000906:Antibodies; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D066246:ErbB Receptors; D001616:beta-Galactosidase