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Fusion tag protein

A fusion tag and fusion protein technology, applied in the field of molecular biology, can solve problems such as expression inhibition

Active Publication Date: 2014-02-05
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is widely expressed in normal tissues, but its expression is significantly suppressed in cancer cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Obtaining of wild-type HREV107 (1-125) gene fragment and construction of C89S mutant

[0034] The DNA fragment of wild-type HREV107 (1-125) can be obtained by PCR amplification reaction using a human liver cDNA library (purchased from Wuhan Sanying Biotechnology Co., Ltd.) as a template.

[0035] Using the human liver cDNA library as a template and using NdeI-REV-N / REV-XhoI-C as primers, PCR amplification was performed to obtain wild-type HREV107(1-125) with NdeI and XhoI restriction sites at both ends. Gene fragment. The fragment was inserted into the pET-21a vector through these two restriction sites to obtain the pET-21a-HREV107N-His plasmid. The primer sequences are as follows:

[0036] NdeI-REV-N: 5'-GGAATTC CATATG CGTGCGCCCATTCCA-3' (SEQ ID No: 4)

[0037] REV-XhoI-C: 5'-CCG CTCGAG GGCGACTCCATAGCGCAG-3' (SEQ ID No: 5)

[0038] Among them, CATATG and CTCGAG are NdeI and XhoI restriction enzyme cutting sites, respectively.

[0039] Since the 89th...

Embodiment 2

[0045] Example 2: Plasmid construction, expression and characterization of Hrevn-rubredoxin-Hrevc-His fusion protein

[0046] In this example, rubredoxin protein was used as the target protein, and it was inserted into the P38-K57 region of HREV107N.

[0047] The rubredoxin protein used in this example and the linker and enzyme cleavage site selected during the construction process are only used to illustrate the construction method of the novel fusion tag protein, and do not limit the scope of application of the present invention.

[0048] 1) Construction of recombinant plasmids

[0049] The recombinant plasmid expression region includes the following 6 parts from A to F from the 5' end to the 3' end, and the sequences are as follows:

[0050] A) HREV107(1-47): refer to the 1st-141th nucleotide sequence of SEQ ID No: 2 in the sequence listing, which encodes the 1st-47th amino acid residues of SEQ ID No: 1;

[0051] B) Linker1:

[0052] 5' ccatgg ggtggttctggtggtggttctggtc...

Embodiment 3

[0067] Example 3: Construction and testing of a novel fusion protein expression system

[0068] In this example, four different proteins were used to test the effect of the novel fusion protein expression system.

[0069] 1) Introduction of the target protein used in the test

[0070] AID: AID (auto-inhibiting domain) is a part of the α subunit of AMP-activated protein kinase (AMPK). AMPK is an important protein kinase, which is expressed in many organs and tissues of the human body, including liver, brain, and skeletal muscle. The increase in the ratio of AMP / ATP in organisms can activate the activity of AMPK. After being activated, AMPK can promote a series of catabolic processes in multicellular organisms, and at the same time inhibit some anabolic pathways, such as the biosynthesis of lipids, proteins and carbohydrates. Thereby maintaining the ratio of AMP to ATP at a constant level. AMPK is therefore a key molecule in bioenergy metabolism.

[0071] C-clamp: Wnt signal...

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Abstract

The invention discloses a fusion tag protein. The fusion tag protein provided by the invention is constructed on the basis of C89S mutant (marked as HREV107N) of wild type HREV107 (1-125) protein, contains HREV107N the amino acid sequence of which is shown as SEQ ID No:1, two Linkers inserted into the P38-K57 random coil region of the mutant protein and multiple cloning sites corresponding short peptide between the two Linkers. The invention further provides a fusion protein expression system, which contains the nucleic acid molecule of the fusion tag protein. Atarget protein gene is inserted into the multiple cloning sites. The C terminal of the fusion tag protein can also be connected to rubredoxin tag protein and His tag so as to facilitate the expression and purification of the fusion protein.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a novel fusion tag protein and a novel fusion protein expression system based on the fusion tag. Background technique [0002] Transferring exogenous genes into host cells such as Escherichia coli for expression to obtain the corresponding target protein is one of the important methods to obtain the desired protein in the process of scientific research and production. Escherichia coli has many advantages as a host cell for protein expression: such as easy reproduction, high protein expression and low price. However, it also has many limitations, for example: some heterologous proteins are expressed in low levels when expressed in E. coli, some proteins are easily degraded after expression, or the expressed foreign proteins are toxic to host cells, resulting in bacterial growth arrest or death. Or although the expression level is high, the protein is aggregat...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C07K19/00C12N15/62C12N15/70
CPCC07K14/47C07K2319/21C07K2319/50
Inventor 夏斌段博陈安琦
Owner PEKING UNIV
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