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Detection of Membrane Protein Interactions by Cell-based Tango Assays

采用基于细胞的Tango测定法检测膜蛋白相互作用

Yan Yan Yan Yan
Ting-Hai Xu Ting-Hai Xu
KH Kaleeckal G. Harikumar
LM Laurence J. Miller
KM Karsten Melcher
HX H. Eric Xu

发布: 2017年11月20日第7卷第22期 DOI: 10.21769/BioProtoc.2903 浏览次数: 11147

评审: Alexandros AlexandratosLokesh KalekarAnonymous reviewer(s)

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Abstract

The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea et al., 2008). Association between the two interaction partners leads to an efficient cleavage of the transcription factor, allowing it to translocate to the nucleus and activate a luciferase reporter gene as measurement of the interactions. In this modified assay, we fused one copy of the membrane-spanning amyloid precursor protein (APP) C99 region to TEV site-rTA (C99-TEV site-rTA) and a second copy to TEV protease (C99-TEV) to analyze intramembrane C99-C99 interaction in live cells.

Keywords: Tango assay (Tango测定法) search γ-Secretase (γ-分泌酶) search Cleavage (裂解) search Activity (活性) search Interaction (相互作用) search C99 (C99) search

Background

The amyloid precursor protein (APP) has three dimerization domains in its N-terminal extracellular domain. In addition, APP can also form dimers through the membrane-bound C99 (C-terminal 99 amino acid fragment) region. Importantly, C99 dimerization has been linked to Aβ production in Alzheimer’s disease (AD) pathology. The Tango assay described here and schematically shown as cartoon in Figure 1 is a fast and sensitive method for investigating homodimerization of C99 and other membrane proteins (Yan et al., 2017).


Figure 1. Cartoon illustration of the Tango interaction assay. Upon membrane cleavage of the C99 hybrid protein by TEV protease, the rTA transactivator protein is released from the membrane into the cytoplasm. This allows rTA to enter the nucleus and bind the tetO DNA-binding site upstream of an integrated luciferase reporter gene to stimulate luciferase reporter gene activity as measured by luminescence. (Yan et al., 2017).

Here we use the Dual-luciferase reporter assay kit. The stably integrated luciferase-Firefly reads represent the γ-secretase cleavage activity, while the transfected Renilla luciferase reads serve as normalization standard.

Materials and Reagents

  1. Pipette tips (VWR)
  2. 24-well plate (Corning, Costar®, catalog number: 3524 )
  3. 96-well plate (Corning, catalog number: 3595 )
  4. Cell culture flask (Corning, catalog number: 430639 )
  5. 96-well OptiPlate (PerkinElmer, catalog number: 6005290 )
  6. Gel loading pipette tip
  7. PS1/PS2-deleted HTL cells (PS1/PS2 gene were deleted by CRISPR/Cas9 from HTL cells [Xu et al., 2016])
  8. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  9. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  10. Trypsin 0.25%-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  11. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
  12. Dual-luciferase reporter assay kit (Promega, catalog number: E1960 )
  13. X-tremeGENE 9 Reagent (Roche Diagnostics, catalog number: 06365787001 )
  14. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: S374-1 )
  15. Potassium phosphate monobasic (KH2PO4) (EMD Millipore, catalog number: PX1565-1 )
  16. Sodium chloride (NaCl) (EMD Millipore, catalog number: SX0420-5 )
  17. Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366-500 )
  18. 10x phosphate buffered saline (PBS buffer) (see Recipes)
  19. 1x passive lysis buffer (PLB) (see Recipes)
  20. LAR2 substrate (see Recipes)
  21. Stop & Glo® Reagent (see Recipes)

Equipment

  1. Micro pipette (Eppendorf)
  2. Vacuum pump
  3. 37 °C, 5% CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 Water-Jacketed)
  4. Cell culture microscope (Nikon Instruments, model: Eclipse TS100 )
  5. Shaker (ARMA LAB, model: Orbital Shaker 100 )
  6. Biosafety cabinet (The Baker, model: SterilGARD® e3 )
  7. EnVision Multilabel Plate Reader (PerkinElmer, model: EnVision Multilabel )

Software

  1. GraphPad Prism 5 (https://www.graphpad.com/scientific-software/prism/)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Yan, Y., Xu, T., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017). Detection of Membrane Protein Interactions by Cell-based Tango Assays. Bio-protocol 7(22): e2903. DOI: 10.21769/BioProtoc.2903.
  2. Yan, Y., Xu, T. H., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017a). Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the gamma-secretase cleavage sites. J Biol Chem, 292: 15826-15837.

    3. ris ris Download Citation in RIS Format

分类

分子生物学 > 蛋白质 > 蛋白质-蛋白质相互作用

生物化学 > 蛋白质 > 表达

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