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- Junjun Gao
- Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, 1700 4th Street, San Francisco, CA 94143; and
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- Sachdev S. Sidhu
- Department of Protein Engineering, Genentech, Inc, 1 DNA Way, South San Francisco, CA 94080
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- James A. Wells
- Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, 1700 4th Street, San Francisco, CA 94143; and
書誌事項
- 公開日
- 2009-03-03
- DOI
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- 10.1073/pnas.0812952106
- 公開者
- Proceedings of the National Academy of Sciences
この論文をさがす
説明
<jats:p> We present a general strategy for identification of conformation-specific antibodies using phage display. Different covalent probes were used to trap caspase-1 into 2 alternative conformations, termed the on-form and the off-form. These conformation-trapped forms of the protease were used as antigens in alternating rounds of selection and antiselection for antibody antigen-binding fragments (Fabs) displayed on phage. After affinity maturation, 2 Fabs were isolated with <jats:italic>K</jats:italic> <jats:sub>D</jats:sub> values ranging from 2 to 5 nM, and each bound to their cognate conformer 20- to 500-fold more tightly than their noncognate conformer. Kinetic analysis of the Fabs indicated that binding was conformation dependent, and that the wild-type caspase-1 sits much closer to the off-form than the on-form. Bivalent IgG forms of the Fabs were used to localize the different states in cells and revealed the activated caspase-1 is concentrated in a central structure in the cytosol, similar to what has been described as the pyroptosome. These studies demonstrate a general strategy for producing conformation-selective antibodies and show their utility for probing the distribution of caspase-1 conformational states in vitro and in cells. </jats:p>
収録刊行物
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- Proceedings of the National Academy of Sciences
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Proceedings of the National Academy of Sciences 106 (9), 3071-3076, 2009-03-03
Proceedings of the National Academy of Sciences