Abstract
Studying protein-protein interaction is crucial to understand the fundamental processes of molecular biology. High-throughput screening, such as immunoprecipitation followed by proteomic analysis, allows for the identification of numerous candidate partners that might interact with a selected protein. However, experimental validation of protein-protein interaction requires conventional cloning and recombinant protein expression/purification, which are complicated and labor-intensive techniques. Here, we demonstrate an efficient experimental pipeline for verifying protein-protein interactions between a bait protein using the example of Odontoglossum ringspot virus (ORSV) capsid protein (CP) and the host CP-binding protein. These candidate CP-binding proteins were identified through high-throughput proteomic and transcriptomic approaches. Using the TOPO cloning strategy, each candidate gene was cloned into an expression vector for the expression of His-tagged recombinant proteins in a single step of an in vitro transcription/translation system. Such expressed His-tagged candidates can be used as prey with the CP bait protein in a co-immunoprecipitation (co-IP) assay to verify their physical interaction. Without the need for traditional protein expression and purification, this pipeline simplifies the validation process and provides a solution for high-throughput protein-protein interaction studies.
Keywords: Protein-protein interaction, in vitro transcription/translation, Co-immunoprecipitation
Materials and Reagents
Equipment
Procedure
Figure 1. Illustration of the in vitro detection of the protein interaction procedure. A. Genes encoding prey proteins are cloned into the pEXP5-CT/TOPO vector using the TOPO cloning system. B. Expression of C-terminal His-tagged recombinant proteins by cell-free in vitro transcription/translation. C. Co-immunoprecipitation is performed to analyze interaction between the His-tagged prey and GST-tagged bait using a bait-specific antibody. An anti-His antibody was used for prey detection and an anti-GST antibody for examining bait precipitation.
Recipes
Acknowledgments
This work was supported by grants from the Ministry of Science and Technology, Taiwan (NSC-102-2313-B-002-068-MY3 and NSC-102-2313-B-002-066-B-MY3) to S.-S. Lin and (NSC-99-2313-B-002-043-MY3) to Y.-C. Chang.
References
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