Protein regulation system as well as preparation method and application thereof

A technology for regulating systems and proteins, applied in the field of bioengineering, can solve the problems of low expression level, low expression range of regulated proteins, and reduce the sensitivity of DDD regulation system, and achieve the effect of large amount of protein, sensitive regulation and high regulation range

Active Publication Date: 2017-11-03
BLUE ELEGANT BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current DDD regulatory system still has defects: (1) the expression level of the protein when regulating the expression of the target protein is lower than the protein expression level when the regulatory sequence is removed; (2) the expression level of the regulating protein is low
[0007] The effects of different connecting peptides are different. Although some connecting peptides can increase the expression of the target protein regulated by the DDD regulatory system, they will also increase the background level of the target protein when no stabilizer is added, but reduce the DDD regulatory system. Sensitivity, so it is necessary to select a suitable connecting peptide to connect the ecDHFR protein and the target protein to be expressed

Method used

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  • Protein regulation system as well as preparation method and application thereof
  • Protein regulation system as well as preparation method and application thereof
  • Protein regulation system as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: Construction of protein regulation system

[0049] Construct the 293T vector, and insert the GPC3 band (EAAAK) into the pEGFP-C1 vector through the restriction sites of AgeI and EcoRI 6 The DDD regulatory fragment of the connecting peptide was inserted to obtain pGPC3-EGFP-(EAAAK) 6 -DHFR-CAR3 vector, obtained vector such as figure 1 shown.

Embodiment 2

[0050] Embodiment 2: Construction of protein regulation system

[0051] Construct the 293T vector, and insert the GPC3 band (EAAAK) into the pEGFP-C1 vector through the restriction sites of AgeI and EcoRI 3 The DDD regulatory fragment of the connecting peptide was inserted to obtain pGPC3-EGFP-(EAAAK) 3 -DHFR-CAR3 vector, obtained vector such as figure 2 shown.

Embodiment 3

[0052] Embodiment 3 connecting peptide adopts A (EAAAK) 5 A substitute

[0053] Construct the 293T vector, in the pEGFP-C1 vector, the GPC3 band A (EAAAK) is passed through the restriction site of AgeI and EcoRI 5 The DDD regulatory fragment of the A-linked peptide is inserted to obtain the pGPC3-EGFP-AEAAAK5A-DHFR-CAR3 vector, and the obtained vector is as follows image 3 shown.

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Abstract

The invention relates to a protein regulation system as well as a preparation method and application thereof. Dihydrofolate reductase in a protein regulation system and to-be-regulated target protein are connected by adopting rigid connecting peptide; the rigid connecting peptide is (EAAAK)n or A(EAAAK)nA, wherein n is an arbitrary integer ranging from 1 to 6, and preferably, n is 6. The protein regulation system disclosed by the invention has higher target protein expression quantity, higher regulation ratio, higher regulation amplitude and very wide application value.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a protein regulation system and its preparation method and application, in particular to a protein regulation system optimized after rigid link peptide connection and its application. Background technique [0002] The DDD (DHFR degradation domain) regulatory system is a regulatory system that uses the ubiquitin protease system to regulate the target protein. It utilizes the fusion of the dihydrofolate reductase (ecDHFR) of Escherichia coli and the target protein by controlling whether the stabilizer is added or not. The target protein is regulated at the protein level. [0003] ecDHFR can be stabilized by the DHFR inhibitor trimethoprim (TMP). When the stabilizer TMP is not added, ecDHFR and its fusion protein will be marked by ubiquitin, and then recognized and degraded by the proteasome, so that the target protein will not be expressed. When the stabilizer TMP is added, TMP will b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A61K48/00A61P35/00
CPCA61K48/0033A61K48/0066C12N15/85C12N2800/107C12N2840/10
Inventor 梁兴祥王美玲郭文中秦莉姚永超陈小平
Owner BLUE ELEGANT BIOTECH CO LTD
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