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Retention effect-enhanced endoplasmic reticulum retention signal peptide mutant

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

Active Publication Date: 2016-02-03
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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  • Retention effect-enhanced endoplasmic reticulum retention signal peptide mutant
  • Retention effect-enhanced endoplasmic reticulum retention signal peptide mutant
  • Retention effect-enhanced endoplasmic reticulum retention signal peptide mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0022] (1) The target gene of Aga2-FLAG-ERS complex containing different retention signal peptides was cloned by PCR method.

[0023] PCR reaction system: 10×KODbuffer, 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; ddH 2 0 to 50 μl.

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

[0025] The designed primer list is as follows:

[0026]

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

Embodiment 2

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

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

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, resuspend the cells with 20 μl of solution B and 0.3 μl of fluorescent antibody Anti-FLAG-APC (GenScript, 0.5 μg / μl), and place the cells at 4 °C for 30 min at room temperature. The whole process is protected from light. The labeled cells were centrifuged at 3000 rpm for 2 min, and the supernatant was removed. Wash once more with solution B, and finally resuspend the cells in 1×PBS. The resuspended cells are used for CytoFLEX Flow cytometry analysis, the detected fluorescent signal channel is APC. Under the same voltage and threshold conditions, according to the strength of the APC signal, it can be judged that the Aga2-FLAG-ERS complex is displaye...

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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 is 4 amino acids, which is KDEL in human cells (Raykhel, I., Alanen, H., Salo, K., Jurv...

Claims

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Application Information

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Patent Type & Authority Applications(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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