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A reflectin extracting method

An extraction method and protein technology, which is applied in the field of extracting reflectin protein, can solve the problems of low solubility and inability to obtain reflectin protein, etc.

Active Publication Date: 2017-12-15
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Similarly, due to the extremely low solubility of reflectin in physiological conditions and in various buffers used in general experiments, other protein extraction methods cannot obtain active soluble form of reflectin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Expression of Reflexin

[0026] The full-length SoRef2 gene (from cuttlefish Sepia officinalis, GenBank ID: HE687200.1) was synthesized by Beijing Biomed Gene Technology Co., Ltd., and cloned into the expression vector pCOLD1 (Takara), with a His fusion tag at its N-terminal, The promoter is cold-inducible promoter cspA, the operon is lacZ, and the expression system is Escherichia coli BL21 (DE3) strain. When the OD600 of the bacteria reached 0.6-0.8, they were induced with IPTG at a final concentration of 20 μM at a temperature of 288K for 20 hours, and then the bacteria were harvested.

Embodiment 2

[0027] Embodiment 2: Purification of reflectin protein

[0028] The harvested bacteria were resuspended in buffer T (20mM Tris, 150mM NaCl, pH 8.0), and placed on ice for ultrasonic lysis. The parameters of ultrasonic lysis were: 200-220w, working time 10s, gap time 5s, cycle 100 times .

[0029] After the lysate was centrifuged at 15000rpm for 30min, the precipitate was retained, and the precipitate was resuspended in buffer R (20mM Tris, 150mM NaCl, 0.5% Triton X-100, pH 8.0), incubated in a rotary shaker at room temperature for 30min, and centrifuged again , to collect the precipitate. The resulting precipitate was washed three times with ultrapure water, then resuspended in buffer S (20 mM Tris, 150 mM NaCl, 0.05% SDS, pH 8.0), and incubated on a rotary shaker at room temperature for 30 min to dissolve the luciferin.

[0030] After centrifugation at 15,000 rpm for 30 min, the supernatant containing the reflectin protein was taken, and imidazole was added thereto to a fin...

Embodiment 3

[0032] Example 3: Further purification and verification of reflectin protein

[0033] In order to further obtain the protein with uniform particles, the particle distribution analysis and separation of the reflectin protein were carried out using a gel filtration chromatography column Superdex 200 10 / 300 GL (GE Healthcare, Sweden), and the mobile phase buffer used was 20mM Tris, 0.1 % SDS, pH 8.0.

[0034] Such as figure 2 As shown, the obtained reflectin protein presents two peaks, which represent two distributions of protein particle size, and the first peak is relatively narrow and symmetrical, indicating that the protein particles distributed here are relatively more uniform. Gel filtration chromatography column analysis is used to separate particles of different sizes, and at the same time achieve the effect of further purification and remove impurity proteins. Comparedfigure 1 and figure 2 According to the electrophoresis gel map, after this step, figure 2 The pro...

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Abstract

The invention belongs to the technical field of biology, and provides a reflectin extracting method. The method includes culturing a bacterial strain expressing reflectin, harvesting bacteria bodies, performing ultrasonic decomposition, performing centrifugation to obtain a precipitate, then resuspending and incubating the precipitate in a plurality of buffer liquids, and performing purification. The reflectin in a soluble form and having activity can be prepared through the method, thus providing beneficial conditions for reflectin property researching and better reflectin utilization.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the extraction of reflectin protein. Background technique [0002] Cephalopods (including octopus, cuttlefish, and squid) can quickly adjust the color of their skin to blend with the environment for concealment, or form a sharp contrast with the environment to alert predators and protect themselves from attack . One of the main reasons for the dynamic color change of cephalopods is the structural color produced by the physical action of light on the ordered microstructures in the cell tissue. The microstructure responsible for this dynamic structural color is made up of a special protein called reflectin. Reflectin is widely distributed in the cellular tissues of cephalopods, and has a variety of morphologies and assembly types. Their optical properties have been extensively studied. However, the structure of this protein and the inherent complexity of the structural color caused by r...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K1/36C07K1/30C07K1/22C07K1/16C12R1/19
CPCC07K14/43504
Inventor 谢灿管哲蔡甜甜
Owner PEKING UNIV
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