| Autor: |
Pin-Chun Lin, Ya-Chun Chang, Shih-Shun Lin |
| Jazyk: |
angličtina |
| Rok vydání: |
2016 |
| Předmět: |
|
| Zdroj: |
Bio-Protocol, Vol 6, Iss 9 (2016) |
| Druh dokumentu: |
article |
| ISSN: |
2331-8325 |
| DOI: |
10.21769/BioProtoc.1800 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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