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-c...

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Bibliographic Details
Published in:	Journal of immunological methods Vol. 303; no. 1; pp. 19 - 39
Main Authors:	Shaki-Loewenstein, Shelly, Zfania, Rahely, Hyland, Stephen, Wels, Winfried S., Benhar, Itai
Format:	Journal Article
Language:	English
Published:	Amsterdam Elsevier B.V 01-08-2005 Elsevier
Subjects:	Animals Antibodies - chemistry Antibodies - genetics beta-Galactosidase - metabolism Biological and medical sciences Cell Line Cell Line, Tumor Cercopithecus aethiops COS Cells Cytoplasmic intrabodies Escherichia coli Fundamental and applied biological sciences. Psychology Fundamental immunology Fusion protein Humans Immunoblotting Immunoglobulin Fragments - biosynthesis Immunoglobulin Fragments - chemistry Immunoglobulin Fragments - genetics Immunoglobulin Variable Region - biosynthesis Immunoglobulin Variable Region - chemistry Immunoglobulin Variable Region - genetics Intracellular Fluid - chemistry Intracellular Fluid - immunology Intracellular Fluid - metabolism Lac Operon Maltose-binding protein Molecular chaperone Molecular immunology Protein Folding Receptor, Epidermal Growth Factor - immunology scFv fragments Techniques PBS HRP Maltose-binding protein EGFP ER EGFR Intrabodies Molecular chaperone ECL mAb scFv fragments AMEF ONPG X-gal scFv Cytoplasmic intrabodies IPTG MBP Fusion protein PCR ELISA Binding protein Chaperone Intracellular Cytoplasmic inlrabodics Single chain antibody Immunological method
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Summary:	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 β-galactosidase enzyme, others that neutralize the wild-type β-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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0022-1759 1872-7905
DOI:	10.1016/j.jim.2005.05.004