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An endoplasmic reticulum retention signal peptide mutant with enhanced retention effect

A signal peptide and endoplasmic reticulum technology, applied in the field of bioengineering, can solve the problems of difficult detection of detection systems, measurement signal amplification, weak measurement signals, etc.

Active Publication Date: 2020-04-03
上海绅道生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the measurement signal generated by the protease hydrolysis reaction at low concentration in the cell is very weak, and it is difficult to detect with the general detection system, so that the analysis of the protease hydrolysis reaction in the cell body faces the problem of measurement signal amplification.

Method used

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  • An endoplasmic reticulum retention signal peptide mutant with enhanced retention effect
  • An endoplasmic reticulum retention signal peptide mutant with enhanced retention effect
  • An endoplasmic reticulum retention signal peptide mutant with enhanced retention effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 The construction method of the genetically engineered bacteria producing Aga2-FLAG-ERS complex

[0022](1) PCR method was used to clone the target gene of Aga2-FLAG-ERS complex containing different retention signal peptides.

[0023] PCR reaction system: 10 × KOD buffer, 5 μl; dNTP (2.5 mM), 5 μl; primer F (10 μM), 2 μl; primer R (10 μM), 2 μl; Pfu polymerase, 0.5 μl; template ( vector pESD), 1 μl; add ddH 2 0 to 50 μl.

[0024] PCR amplification system: 94 °C, 5 min; 94 °C, 30 s, 55 °C, 30 s, 72 °C, 30 s, 25 cycles; 72 °C, 5 min; 4 °C, ∞.

[0025] The designed primer list is as follows:

[0026]

[0027] (2) After the obtained PCR product was recovered with 1.0% agarose gel, it was double-digested with restriction endonucleases PstI and EcoRI. The digestion system was: PstI, 0.5 μl; EcoRI, 0.5 μl; 10 × O buffer, 5 μl; PCR recovered product, 30 μl, add ddH 2 0 to 50 μl. After treatment at 37°C for 6 h, it was recovered with 1% agarose gel, and then en...

Embodiment 2

[0029] Example 2 Induced expression of endoplasmic reticulum retention signal peptide mutant Aga2-FLAG-ERS complex

[0030] Saccharomyces cerevisiae EBY100 containing plasmids with different retention signal peptides were inoculated in YNB-CAA-Glucose medium and cultured at 30 °C and 225 rpm for 12 h. Cultivate to OD600=2~3, change YNB-CAA-Galactose medium to induce and make the initial OD600=0.5, culture at 30 ℃, 225 rpm, and take samples after induction 3h and 6h.

Embodiment 3

[0031] Example 3 Application of Flow Cytometry to Detect the Retention Effect of Different Retention Signal Peptides

[0032] 10 per sample 6 Cells were washed once with solution A and then once with solution B. The whole process should be operated on ice, with a rotation speed of 3000rpm and centrifugation for 2min. Finally, 20 μl of solution B and 0.3 μl of fluorescent antibody Anti-FLAG-APC (GenScript, 0.5 μg / μl) were used to resuspend the cells, and the cells were placed at 4°C for min and then at room temperature for 30 min, and the whole process was protected from light. The labeled cells were centrifuged at 3000rpm for 2min, and the supernatant was discarded. Wash once more with solution B, and finally resuspend the cells with 1×PBS. The resuspended cells are used for CytoFLEX flow cytometry analysis, and the fluorescent signal channel detected is APC. Under the same voltage and threshold conditions, according to the strength of the APC signal, the Aga2-FLAG-ERS comp...

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Abstract

The invention discloses a retention effect-enhanced endoplasmic reticulum retention signal peptide mutant WEHDEL, which belongs to the technical field of bioengineering. Through mutated yeast endoplasmic reticulum retention signal peptide FEHDEL, a series of retention signal peptide mutants are obtained. By constructing a pESD-Aga2-FLAG-ERS recombinant plasmid (ERS, ER sequestration signal, endoplasmic reticulum retention signal peptide), the induction and surface display conditions of an Aga2-FLAG-ERS complex in yeast are analyzed, and the fluorescence labeling technique and a flow cytometer are utilized to detect the protein displayed on the surfaces of cells. According to the intensity of cell surface fluorescence, the intensity of the retention effect of the endoplasmic reticulum retention signal peptide is analyzed. The analysis of the flow cytometer finds that the retention effect of the retention signal peptide mutant WEHDEL is remarkably enhanced in comparison with the retention effect of the original FEHDEL. In a recently established yeast endoplasmic reticulum retention system, the retention effect of the endoplasmic reticulum retention signal peptide is utilized to prolong the retention time of a protein substrate in an endoplasmic reticulum in order to significantly increase the concentration of the substrate in the endoplasmic reticulum, so that protease still can generate a strong hydrolysis reaction signal even under low concentration and low activity state, and thereby detection and analysis are facilitated.

Description

technical field [0001] The invention relates to an endoplasmic reticulum retention signal peptide mutant with enhanced retention effect, belonging to the technical field of bioengineering. Background technique [0002] The endoplasmic reticulum retention signal peptide is a polypeptide sequence of about 4 amino acids in length at the carboxy-terminal of the protein. It can interact with the protein on the inner membrane of the endoplasmic reticulum, and make the protein carrying the retention signal peptide shuttle back and forth between the endoplasmic reticulum and the Golgi apparatus to prolong the retention time of the protein in the endoplasmic reticulum and the Golgi apparatus, so as to be fully modified Or degradation, thereby affecting the correct folding and secretion of proteins. Early studies believed that the length of the retention signal peptide in the endoplasmic reticulum was 4 amino acids, which was KDEL in human cells (Raykhel, I., Alanen, H., Salo, K., Ju...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C12N15/81C12N1/19C12R1/865
Inventor 易犁梅萌张桂敏马立新马延和
Owner 上海绅道生物科技有限公司
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