Preparation method of reversible photochromic biliprotein

A technology of bilirubin and photochromism, which is applied in the field of pigment protein materials in biotechnology, and can solve problems such as application limitations

Inactive Publication Date: 2003-09-24
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method has limited applications in the fields of food, cosme...

Method used

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  • Preparation method of reversible photochromic biliprotein

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Embodiment 1

[0017] (1) The aphA numbered AB028873 in GenBank was cloned into the expression vector pET30 of Novagen Company, and the recombinant expression vector was transformed into Escherichia coli to obtain the corresponding engineering bacteria of Escherichia coli, which was used to produce AphA. The AphA produced in this way has an affinity tag composed of 6 histidines. Therefore, a metal ion chelating affinity chromatography column matching the affinity tag can be used to separate and purify AphA.

[0018] (2) Mix the apoprotein AphA obtained in step (1) with PC, and react according to the following reaction conditions: reaction temperature 37°C, pH value 7.5; cofactors required for the reaction: ①5mmol / L magnesium ion, ②5mmol / L L of mercaptoethanol.

[0019] The reaction results in the phytochrome reversible photochromic bilirin protein AphA.

Embodiment 2

[0021] (1) The aphA numbered AB028873 in GenBank was cloned into the expression vector pGEMEX of Novagen Company, and the recombinant expression vector was transformed into Escherichia coli to obtain the corresponding E. coli engineering bacteria, which was used to produce AphA.

[0022] (2) Mix the apoprotein AphA obtained in step (1) with PC, and react according to the following reaction conditions: reaction temperature 37°C, pH value 7.5; cofactors required for the reaction: ①5mmol / L magnesium ion, ②5mmol / L L of mercaptoethanol.

[0023] The reaction results in the phytochrome reversible photochromic bilirin protein AphA.

Embodiment 3

[0025] (1) The cphl numbered D64001 in GenBank was cloned into the expression vector pET30 of Novagen Company, and the recombinant expression vector was transformed into Escherichia coli to obtain the corresponding E. coli engineering bacteria, which was used to produce Cphl. The Cphl produced in this way has an affinity tag composed of 6 histidines, therefore, a metal ion chelating affinity chromatography column matching the affinity tag can be used to separate and purify Cphl.

[0026] (2) Mix the apoprotein Cphl obtained in step (1) with PC, and react according to the following reaction conditions: reaction temperature 37°C, pH value 7.5; cofactors required for the reaction: ① 5mmol / L magnesium ion, ② 5mmol / L L of mercaptoethanol.

[0027] The reaction results in the phytochrome reversible photochromic bilirin protein Cphl.

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Abstract

The present invention discloses a preparation method of reversible photochromic biliprotein, and is characterized by that it utilizes gene engineering technique to express the apoprotein of photochrome reversible photochromic biliprotein and phycocyanin lyase lyzed from phycocyanin by correspondently catalyzing polycocyanobilin, then uses the lyase or photochrome aproprotein to catalyze phycocyanobilin to make it transfer from phycocyanin and its subunit, and at the same time externally recombined with biliprotein and apoprotein so as to prepare the reversible photochromic biliprotein. Said invention does not use organic solvent and detergent, only uses protein and aqueous solution. It is suitable for food, health-care and medicine function fields.

Description

technical field [0001] The invention belongs to the field of pigment protein materials in biotechnology, and in particular relates to a preparation method of reversible photochromic bilirin protein. technical background [0002] Phytochromes of the biliprotein class are functional pigment proteins present in plants. According to their absorption spectrum characteristics and physiological functions, phytochromes can be divided into five categories: phytochrome A, phytochrome B, phytochrome C, phytochrome D, and phytochrome E. The bacterial phytochrome (cyanobacterial phytochrome) discovered so far mainly includes bacterial phytochrome 1 (cyanobacterial phytochrome 1, referred to as Cphl) and bacterial phytochrome A (cyanobacterial phytochrome A, referred to as AphA). Bile pigment (bilin) ​​covalently binds to the sulfhydryl group of phytochrome apoprotein through thioether bond, and its type and its interaction with apoprotein determine the spectral properties of phytochrome...

Claims

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Application Information

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IPC IPC(8): C07K14/405C07K14/415C12N15/29C12N15/70
Inventor 赵开弘冉勇董依然
Owner HUAZHONG UNIV OF SCI & TECH
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