检测蛋白质相互作用的体外转录/翻译系统

Pin-Chun Lin; Ya-Chun Chang; Shih-Shun Lin

doi:10.21769/BioProtoc.1800

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Peer-reviewed

An in vitro Transcription/translation System for Detection of Protein Interaction

检测蛋白质相互作用的体外转录/翻译系统

PL Pin-Chun Lin

YC Ya-Chun Chang

SL Shih-Shun Lin email

发布: 2016年05月05日第6卷第9期 DOI: 10.21769/BioProtoc.1800 浏览次数: 16747

评审: Zhaohui LiuPablo Bolanos-VillegasAnonymous reviewer(s)

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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 (蛋白质-蛋白质相互作用)

in vitro transcription/translation (体外转录/翻译)

Co-immunoprecipitation (免疫共沉淀)

Materials and Reagents

TOPO cloning for the protein expression construct
1. Heat shock-competent cell of E. coli strain DH5α
2. pEXP5-CT/TOPO TA Expression Kit (Thermo Fisher Scientific, Invitrogen^TM, catalog number: V960-06 )
3. Forward/reversed primers for candidate genes
4. ExTaq polymerase (Takara Bio Company, catalog number: RR001A )
5. UltraPure agarose (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 16500 )
6. QIAEX^® II Gel Extraction Kit (QIAGEN, catalog number: 20051 )
7. Luria-Bertani (LB) broth and LB agar plates
8. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A0166 )
9. T7 forward primer (5’-TAATACGACTCACTATAGGG-3’)
10. Presto^TM Mini Plasmid Kit (Geneaid Biotech Ltd., catalog number: PDH300 )
Expression of recombinant protein via single-step in vitro transcription/translation
1. Expressway^TM Cell-Free E. coli Expression System (Thermo Fisher Scientific, Invitrogen^TM, catalog number: K9901-00 )
2. Polyacrylamide gel electrophoresis (PAGE) system (Bio-Rad Laboratories, catalog number: 1610158 )
3. Anti-His (C-term)-HRP monoclonal antibody (Thermo Fisher Scientific, Invitrogen^TM, catalog number: R931-25 )
4. COOMASSIE Brilliant Blue G-250 (VWR International, J.T.Baker, catalog number: F789 )
Protein-protein interaction and co-immunoprecipitation (co-IP)
1. pGEX-4T-1 DNA vector (GE Healthcare, catalog number: 28-9545-49 )
2. Bait protein with tag [herein, GST-CP (recombinant of ORSV CP protein with the N-terminus fused to glutathione S-transferase)
3. Anti-bait antibody [herein, anti-CP antibody (Lee and Chang, 2008)]
4. Protein G PLUS-Agarose (Santa Cruz Biotechnology, catalog number: sc-2002 )
5. Anti-tag antibody (anti-GST mouse monoclonal antibody) (Bioman, catalog number: GST001M )
6. Tris base (J.T.Baker, catalog number: 4109 )
7. NaCl (Sigma-Aldrich, catalog number: S9888 )
8. Glycerol (Sigma-Aldrich, catalog number: G5516 )
9. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
10. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
11. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
12. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
13. Acrylamide/bis-acrylamide (37.5:1) (Bio-Rad Laboratories, catalog number: 1610158 )
14. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678 )
15. TEMED (Sigma-Aldrich, catalog number: T9281 )
16. Glycine (Sigma-Aldrich, catalog number: G8898 )
17. Methanol (Sigma-Aldrich, catalog number: 322415 )
18. Co-precipitation buffer (see Recipes)
19. Wash buffer (see Recipes)
20. 2x sample buffer (see Recipes)
21. SDS-PAGE separating gel (see Recipes)
22. SDS-PAGE stacking gel (see Recipes)
23. PAGE running buffer (see Recipes)
24. Western blot transfer buffer (see Recipes)

Equipment

Thermal cycler (Biocompare, Biometra, catalog number: T3000 )
Horizontal gel electrophoresis device (Bio-Rad Laboratories)
42 °C waterbath (FIRSTEK, catalog number: B101 )
37 °C Orbital Shaking Incubator (FIRSTEK, catalog number: S300R )
Polyacrylamide gel electrophoresis system (Bio-Rad Laboratories, catalog number: 1658003 )
Western blot transfer apparatus (Bio-Rad Laboratories, catalog number: 1703930 )
Mixer for Eppendorf tube (ELMI North America, catalog number: RM-2L )
Microcentrifuge (Eppendorf AG, catalog number: 05400002 )

Procedure

English

中文翻译

文章信息

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如何引用

Lin, P., Chang, Y. and Lin, S. (2016). An in vitro Transcription/translation System for Detection of Protein Interaction. Bio-protocol 6(9): e1800. DOI: 10.21769/BioProtoc.1800.