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Establishment and expression of fusion protein having function of adsorbing heavy metal ions, and application fusion protein in bioremediation

A technology of fusion protein and metal, applied in the field of fusion protein, can solve the problems of insoluble expression, small molecular weight of metallothionein, and many cysteine ​​residues

Active Publication Date: 2013-09-11
GUANGZHOU BIO ZONE BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Cysteine-rich metallothionein plays an important role in the detoxification of heavy metals, however, it is difficult to perform large-scale recombinant expression in the host using genetic engineering methods
On the one hand, metallothionein has a small molecular weight and many cysteine ​​residues, making it difficult to express solublely; on the other hand, a large number of sulfhydryl groups can also hinder the normal redox pathway of host cells, seriously affecting the normal physiological activities of host cells

Method used

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  • Establishment and expression of fusion protein having function of adsorbing heavy metal ions, and application fusion protein in bioremediation
  • Establishment and expression of fusion protein having function of adsorbing heavy metal ions, and application fusion protein in bioremediation
  • Establishment and expression of fusion protein having function of adsorbing heavy metal ions, and application fusion protein in bioremediation

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Embodiment 1: Construction of recombinant food-grade lactic acid bacteria expression vector pL3UA-Dec-H-R

[0063]Escherichia coli (Escherichia coli) NovaBlue (purchased from Novagen, Inc., Madison, Wisconsin) containing the pL3UA plasmid was placed on a Luria-broth (LB) solid culture plate containing erythromycin antibiotics (ingredients: based on the total weight of the medium, 1 % by weight of broth, 0.5% by weight of yeast extract, 1% by weight of sodium chloride, 1.5% by weight of agar powder, and erythromycin at a final concentration of 100 μg / ml) for streak culture. After 14 hours of anaerobic culture at 37°C, pick a single colony from the plate medium and inoculate it into LB liquid medium containing erythromycin antibiotics (ingredients: 1% broth, 0.5% yeast extract, 1% sodium chloride, final erythromycin at a concentration of 100 μg / ml), cultured statically at 37°C until OD 600 = 0.6. The bacteria were collected by centrifugation and the expression plasmid p...

Embodiment 2

[0065] Embodiment 2: the preparation of recombinant lactic acid bacteria and the expression of fusion protein

[0066] Inoculate the recombinant lactic acid bacteria containing the sequenced correct expression plasmid pL3UA-Dec-H-R into the expression medium containing erythromycin (final concentration is 100ng / ml), (ingredients: based on the total weight of the medium, 1 wt% meat peptone, 1 % by weight beef extract, 0.5% by weight of yeast extract, 2% by weight of glucose, 0.5% by weight of sodium acetate, 0.1% by weight of Tween 80, 0.2% by weight of potassium phosphate, 0.2% by weight of ammonium citrate, 0.01% by weight of magnesium sulfate and 0.0005 weight % manganese sulfate, pH = 6.5), 37° C., anaerobic stationary culture for 1 day. After the fermentation, the cells were collected by centrifugation at 12000 rpm, and 0.02 mol / L phosphate buffer (pH=7.0) was added to resuspend the cells at a ratio of 1:10 (v / w). After the cells were disrupted by an ultrasonic disruptor,...

Embodiment 3

[0067] Example 3: Analysis of the Toxic Heavy Metal Ion Binding Ability of the Metal-binding Polypeptide and the Decamer of the Connecting Peptide and the Fusion Protein of the Skin Cell Adhesion Tripeptide

[0068] In the expression medium (ingredients: based on the total weight of the medium, 1% by weight of meat peptone, 1% by weight of beef extract, 0.5% by weight of yeast extract, 2% by weight of glucose, 0.5% by weight of sodium acetate, 0.1% by weight of Tween 80 , 0.2% by weight of potassium phosphate, 0.2% by weight of ammonium citrate, 0.01% by weight of magnesium sulfate and 0.0005% by weight of manganese sulfate, pH=6.5) add different concentrations of cadmium chloride (CdCl 2 , 200μM, 300μM, 400μM, 500μM), lead acetate (PbAc 2 , 0.2mM, 0.3mM, 0.5mM, 1.0mM) or mercury dichloride (HgCl 2 , 15 μM, 50 μM, 150 μM, 450 μM), and then inoculate lactic acid engineered bacteria expressing the fusion protein of the metal-binding polypeptide and the decamer of the connecting...

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Abstract

The invention discloses a preparation method of a fusion protein (SEQ ID No: 4) of a decamer containing a metal binding polypeptide and a connecting peptide, and a skin cell adhesion tripeptide, and an application of the fusion protein, especially an application of the fusion protein in the field of cosmetics. The preparation method disclosed by the invention is characterized by comprising the following steps of: obtaining a DNA sequence (SEQ ID No: 8) for coding the fusion protein of the decamer containing the metal binding polypeptide and the connecting peptide, and the skin cell adhesion tripeptide by virtue of a gene engineering technique, and then performing recombination expression by a surface display method in lactic acid bacteria. As the metal binding polypeptide and the skin cell adhesion protein have the abilities of binding harmful heavy metal ions and increasing the adhesive skin of an expression host, respectively, the cosmetics prepared from lactic acid engineering bacteria of the fusion protein of the decamer containing the metal binding polypeptide and the connecting peptide and the skin cell adhesion tripeptide expressed by the surface display method are capable of effectively reducing accumulation of toxic heavy metals in skin; and therefore, the effect of bioremediation is achieved.

Description

technical field [0001] The invention relates to a fusion protein of a decamer comprising a metal-binding polypeptide and a connecting peptide and a skin cell adhesion tripeptide, and also relates to a lactic acid bacterium displaying and expressing the fusion protein on the cell surface. Specifically, the amino acid sequence of the fusion protein is shown in SEQ ID No:4. The present invention also relates to the application of the fusion protein or the lactic acid bacteria displaying and expressing the fusion protein on the cell surface in the preparation of cosmetics or medicines for binding toxic heavy metals on the skin surface. In addition, the present invention also relates to a drug or cosmetic for removing toxic heavy metals from the skin surface, the drug or cosmetic comprising the fusion protein or lactic acid bacteria displaying and expressing the fusion protein on the cell surface. Background technique [0002] With the development of industrialization of various...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/74C12N1/21A61K8/64A61K8/99A61K35/74A61K38/16A61K47/48A61P39/02C12R1/25C12R1/245C12R1/01A61K35/744
Inventor 王宏刘斌杨海涛伍建军张绍伟高社
Owner GUANGZHOU BIO ZONE BIOTECH
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