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Construction method of split-TEV-Fast system and application of split-TEV-Fast system in detecting protein interaction

A split-tev-fast and tev-fast technology, applied in the fields of biotechnology and molecular biology, can solve the problems of low reaction sensitivity, inability to accurately quantify the strength of protein interaction, and large interaction space between enzymes and substrates. The effect of sensitivity

Active Publication Date: 2018-05-18
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Split-TEV used wild-type TEV protease in the past. Under the interaction of the target protein to be tested, the complete TEV protease only retains 40% of its original activity and plays a role in the cytoplasm. The reaction space is large and the concentration is relatively low, resulting in low reaction sensitivity, which makes it impossible to accurately quantify the protein interaction strength

Method used

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  • Construction method of split-TEV-Fast system and application of split-TEV-Fast system in detecting protein interaction
  • Construction method of split-TEV-Fast system and application of split-TEV-Fast system in detecting protein interaction
  • Construction method of split-TEV-Fast system and application of split-TEV-Fast system in detecting protein interaction

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Construction process of WEHDEL recombinant vector containing retention signal peptide

[0032] (1) Firstly, the Aga2-FLAG-ENLYFQS-HA-ERS (WEHDEL) target gene was amplified by polymerase chain reaction (PCR). The reaction system is as follows: 50μL system, 10×KOD buffer, 5μL; dNTP (2.5mM) , 3μL; Primer F1 (10μM), 2μL; Primer R1 (10μM), 2μL; Pfu polymerase, 1μL; Template (containing TEV protease substrate polypeptide sequence), 0.5μL (20ng / μL); add double distilled water to 50μL .

[0033] PCR amplification conditions: 95℃, 5min; 95℃, 30s, 57℃, 30s, 72℃, 30s, 25 cycles; 72℃, 5min; 4℃, ∞.

[0034] The primer design is as follows:

[0035] F1 is SEQ ID NO.1

[0036] R1 is SEQ ID NO. 2

[0037] (2) The target fragments were recovered by agarose gel, which were digested with XhoI and EcoRI at 37°C for 6 hours, and then recovered with agarose gel. Double digestion system: target fragment DNA, 30μL; Buffer Cutsmart, 5μL; XhoI, EcoRI, 2μL each; add double distilled water to 50μL.

[0038...

Embodiment 2

[0061] Induced expression of recombinant vector containing Split-TEV-Fast system

[0062] The reagents used to transform the final vector into Saccharomyces cerevisiae EBY200 transformation are: EBY200 competent, 20μL; recombinant plasmid, 1.5μL; Single strand carrier DNA, 25μL; 1M LiAC, 36μL; PEG4000 (50%W / V), 240μL; mix After the system, 30 ℃ water bath for 30 min, transfer to 42 ℃ heat shock for 20 min, centrifugation to collect the bacteria, add 1mL YPD liquid medium to 30 ℃ shaker culture for 1.5 hours, wash the cells with ultrapure water once, then apply to YNB-CAA- Glucose solid medium, 30℃ incubator for 2-3 days. The composition of YNB-CAA-Glucose solid medium is 20g / L glucose, 6.7g / L YNB, 5.4g / L Na 2 HPO 4 ,8.6g / L NaH 2 PO 4 ·H 2 O, 5g / L casamino acids, pH7.4, 15g / L agar.

[0063] Inoculate a single colony in the liquid medium YNB-CAA-Glucose, culture it in a shaker at 30°C, 230rpm until OD600=3, transfer to YNB-CAA-Galactose liquid medium, cultivate it in a shaker at 30°...

Embodiment 3

[0065] Use flow cytometry to detect the results of the Split-TEV-Fast system to detect protein interactions

[0066] A complex composed of Yael, Lto1, and Rli1 was selected to verify the Split-TEV-Fast system, where Lto1 and Yael interact with each other, but not with Rli1. Therefore, Yae1 and Lto1 are set as the experimental group, and Rli1 and Lto1 are the negative control groups. Due to the interaction between Yae1 and Lto1, the split-TEV-Fast -NH2 end and -COOH end are reconstituted to form a complete and active TEV- Fast is to cleave the substrate, and the yeast surface displays Aga2-FLAG-ENLYFQ, and then the single fluorescent signal iFluor 647 is detected by the Anti-FLAG-iFluor 647 fluorescent antibody label. When the target protein is Rli1 and Lto1, if the two do not interact, a complete and active TEV-Fast protease cannot be formed, and -ENLYFQS cannot be cleaved. The yeast surface displays Aga2-FLAG-ENLYFQS-HA Then, the dual fluorescence signal (iFluor 647 and FITC) w...

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Abstract

The invention discloses a method for constructing a split-TEV-Fast system based on saccharomyces cerevisiae and an application of the split-TEV-Fast system in detecting a protein interaction. The method comprises the following steps: 1) constructing a vector pESD-PPI, wherein the vector comprises a forward promoter and a bidirectional promoter induced by galactose, the forward promoter is used forexpressing a TEV-Fast protease substrate as well as FLAG and HA tag sequences, and the bidirectional promoter is used for expressing resolving TEV-Fast protease and a to-be-detected target protein; 2) simultaneously inducing the promoters via the galactose, and inducing equivalent expression of a downstream gene; 3) marking cells via fluorescent antibodies which recognize the FLAG and HA tag sequences; and 4) detecting fluorescence signals on the surfaces of the cells via a flow cytometry, and judging the intensity of the protein interaction in accordance with the single and double fluorescence signals. The method provided by the invention, by adopting endoplasmic reticulum retention signal peptide differing in intensity at -COOH terminals of the TEV-Fast protease and the substrate thereof, can widen a range of detecting the protein interaction; and the intensity of the protein interaction can be analyzed depending on a finally detected proportion of single and double fluorescence signal intensities.

Description

Technical field [0001] The invention belongs to the technical field of biotechnology and molecular biology, and particularly relates to a method for constructing a split-TEV-Fast system and its application in detecting protein-protein interaction. Background technique [0002] The study of protein-protein interaction has extremely important value in many biological fields. In organisms, a protein usually interacts with other proteins to further perform its function, which is the main way of biological information regulation. Protein-protein interaction clarifies the physiological and biochemical reactions and molecular mechanisms in the entire cell, and even the cellular The whole life process. At present, methods for verifying protein-protein interactions mainly include several classic experimental techniques: yeast two-hybrid technique, co-immunoprecipitation technique (Co-IP), GST pull down technique and so on. Yeast two-hybrid technology is characterized by sensitivity, hig...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/62G01N33/68
CPCC07K2319/035C07K2319/60C12N9/50C12N15/81C12N2830/34G01N33/6845
Inventor 易犁王婷张桂敏梅萌王钦宏
Owner HUBEI UNIV
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