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The use of phage display to generate conformation-sensor recombinant antibodies

Abstract

We describe a phage display approach that we have previously used to generate conformation-sensor antibodies that specifically recognize and stabilize the oxidized, inactive conformation of protein tyrosine phosphatase 1B (PTP1B). We use a solution-based panning and screening strategy conducted in the presence of reduced active PTP1B, which enriches antibodies to epitopes unique to the oxidized form while excluding antibodies that recognize epitopes common to oxidized and reduced forms of PTP1B. This strategy avoids conventional solid-phase immobilization owing to its inherent potential for denaturation of the antigen. In addition, a functional screening strategy selects single-chain variable fragments (scFvs) directly for their capacity for both specific binding and stabilization of the target enzyme in its inactive conformation. These conformation-specific scFvs illustrate that stabilization of oxidized PTP1B is an effective strategy to inhibit PTP1B function; it is possible that this approach may be applicable to the protein tyrosine phosphatase (PTP) family as a whole. With this protocol, isolation and characterization of specific scFvs from immune responsive animals should take 6 weeks.

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Figure 1: Generation of single-chain variable fragments (scFvs) for PTP1B-OX.
Figure 2: Site-specific biotinylation of PTP1B (wild-type and CASA mutant).
Figure 3: In-solution panning to enrich PTP1B-OX-specific scFvs.
Figure 4: Screening of scFvs specific to PTP1B-OX.

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References

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Acknowledgements

We thank D. Barford and A. Salmeen at the Institute of Cancer Research, UK, for conducting the structural studies of PTP1B-OX and PTP1B-CASA, and J. Andersen at Cold Spring Harbor Laboratory for purifying the PTP1B-CASA mutant. We thank C.F. Barbas III (Scripps Research Institute) for generously providing us with the pComb3XSS phagemid vector. This work was supported by grants CA53840 and GM55989 from the US National Institutes of Health to N.K.T.

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A.H. and N.K.T. designed the experiments and analyzed the data. A.H. performed the experiments. A.H. and N.K.T. wrote the manuscript. N.K.T. directed the study and obtained grant support. The authors read and approved the final version of the manuscript.

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Correspondence to Nicholas K Tonks.

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Haque, A., Tonks, N. The use of phage display to generate conformation-sensor recombinant antibodies. Nat Protoc 7, 2127–2143 (2012). https://doi.org/10.1038/nprot.2012.132

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