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Hydrophilic domain protein gene

A gene and protein protection technology, which is applied in the application field of protecting enzyme activity and reducing enzyme aggregation, can solve the problems of unknown and unclear anti-stress protection mechanism of hydrophilic proteins, and achieve the effect of preventing structural changes.

Inactive Publication Date: 2016-11-16
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of stress resistance protection of hydrophilic proteins is still unclear
[0003] The genome of Deinococcus radiodurans R1 contains a class of hydrophilic proteins, including a gene encoding a drying stress-related gene, but there is no research report on the function of this gene in protecting enzyme activity and reducing enzyme aggregation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Analysis of Hydrophilic Domain of Dhp Protein HD

[0044] The remarkable feature of the Dhp protein is that it contains a hydrophilic domain (HD), which has a hydrophilic amino acid content of 63.9%. The HD domain contains 8 motifs consisting of 11 amino acid residues. Between the two motifs There are connecting peptides between them, and there are 7 connecting peptides in total, 4 of which are composed of 11 amino acid residues and 3 of which are composed of 7 amino acid residues. Such as figure 1 As shown, the motif analysis of 11 amino acid residues found that the amino acids at the 3rd, 4th, 6th, 7th, 8th, 10th and 11th positions are hydrophilic amino acids (hydrophilic amino acid content is 61.36%) , the amino acids at other positions are hydrophobic amino acids. The motif sequence is characterized by [ΦΦE / QXΦKE / QKΦXE / D / Q], where Φ is a hydrophobic amino acid. For the connecting peptide analysis of 11 amino acids, it was found (55.38% of the hydrophili...

Embodiment 2

[0045] Example 2 Construction of HD hydrophilic domain protein expression vector

[0046] In order to study the protective function of the HD hydrophilic domain, this study analyzed the HD domain, recombined the domains and constructed expression vectors respectively. According to the positions of the 8 motifs composed of 11 amino acids in the Dhp protein, we Divide the Dhp protein into image 3Structures shown: core protein HD (amino acid residue sequence 104-263), and recombinant protein 2HD (containing twice the core protein HD amino acid sequence).

[0047] Using the recombinant plasmid pET28a-dhp stored in our laboratory as a template, add PCR-specific primers to amplify the complete nucleotide sequence: PCR reaction conditions: 98°C for 5min, [98°C for 30sec, 64°C for 30sec, 72°C for 1min] After 33 cycles, 72°C for 10 min, the PCR products were gel-recovered. The DNA product and pET28a vector were digested with EcoRI / NdeI and ligated overnight at 4°C or 16°C. Take 10 ...

Embodiment 3

[0048] Example 3 Induced expression and purification of HD hydrophilic domain protein in Escherichia coli

[0049] (1) Experimental method

[0050] 1. IPTG induces HD hydrophilic domain protein expression

[0051] 1) Streak activation of the successfully constructed recombinant plasmids BL-hd and BL-2hd strains. Pick a single colony and place it in 3 mL of fresh LB medium (containing kanamycin 50 mg / mL), and cultivate overnight at 37° C. and 220 rpm with shaking.

[0052] 2) The next day, the seed solution was transferred to 20mL LB medium containing Km (50mg / mL) at a ratio of 1:100, and cultured with shaking at 37°C and 220rmp, OD 600 About 0.6-0.8.

[0053] 3) Adding IPTG with final concentrations of 0.5, 1.0, and 1.5 mM respectively at 16° C. overnight (without adding IPTG as a blank control) to induce Escherichia coli to perform exogenous expression.

[0054] 4) Take 2 mL of the induced bacterial solution, centrifuge at 5,000 rpm for 5 minutes, and collect the bacteria...

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Abstract

The present invention finds the protective effect of the hydrophilic domain on the enzyme in the Dhp(DR1172) protein. The present invention expresses a core protein containing a complete hydrophilic domain. The experimental results showed that the protein was repeatedly frozen and thawed in liquid nitrogen and H 2 O 2 Both can protect the activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) under shock stress conditions, and both can reduce the aggregation of LDH and prevent its structure from changing to protect its activity.

Description

technical field [0001] The invention relates to a hydrophilic domain protein gene and the protein coded by the gene, and also relates to the application of the gene and protein in protecting enzyme activity and reducing enzyme aggregation. Background technique [0002] Stresses such as drought, salinity, freezing and oxidation are abiotic stress factors that restrict the growth and development of organisms. As an important part of extreme response to adversity stress, highly hydrophilic protein has become a research hotspot, and this protein may act as a molecular chaperone to protect cells under adversity stress. However, the mechanism of protection against stress of hydrophilic proteins is still unclear. [0003] The genome of Deinococcus radiodurans R1 contains a class of hydrophilic proteins, including a gene encoding a drying stress-related gene, but there is no research report on the function of this gene in protecting enzyme activity and reducing enzyme aggregation. ...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N9/96
CPCC07K14/195C12N9/96
Inventor 王劲林敏平淑珍左开井刘盈盈
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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