A diblock fusion protein with dual functions of adhesion and antifreeze, its synthesis method and application

A fusion protein and synthesis method technology, applied in the biological field, can solve the problems of cumbersome operation, cumbersome preparation method of superhydrophobic material, protein inactivation, etc., and achieves the effects of low equipment requirements, easy later production expansion, and low cost.

Active Publication Date: 2022-03-15
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, the research on anti-icing materials has mainly focused on the use of hydrophobic or superhydrophobic surfaces, but the anti-icing effect of hydrophobic materials is not ideal, and the preparation methods of superhydrophobic materials are cumbersome.
There are also a few scientists who have combined antifreeze proteins with polymers to modify the surface of materials, and have achieved good anti-icing effects. However, combining antifreeze proteins with polymers requires modification of reactive groups on the protein, which can easily lead to protein loss. live, and the operation is cumbersome

Method used

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  • A diblock fusion protein with dual functions of adhesion and antifreeze, its synthesis method and application
  • A diblock fusion protein with dual functions of adhesion and antifreeze, its synthesis method and application
  • A diblock fusion protein with dual functions of adhesion and antifreeze, its synthesis method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Recombinant expression vector and strain

[0040] 1 Recombinant expression vector

[0041] The expression of fusion protein MP-AFP is configured to connect to a polypeptide to connect to a polypeptide to connect to a polypeptide. The 5 'end of the gene sequence is added to the BamHI restriction enzyme digestion site and the NOTI restriction enzyme disaster is added; the manual synthesis of the gene sequence and cloned into E. coli expression vector PET-28A to obtain recombinant expression vectors PET-28A-MP-AFP containing T7 strong label, LCA lactose, kanamycin resistance marker site, and hexamemonic label, such as the recombinant expression vector. figure 1 Indicated.

[0042] 2 chemical transformation of E. coli

[0043] The substance E. coli BL21 (DE3) is melted on ice. The correct recombinant expression vector PET-28A-MK-AFP will be constructed, and the expression vector of the expression tyrosinase is mixed with the sensitudinal E. coli BL21 (DE3), incubated...

Embodiment 2

[0044] Example 2: Expression purification of fusion protein MP-AFP

[0045] 1 Escherichia coli fermentation expression fusion protein MP-AFP

[0046] E. coli converted into transformed E. coli is activated on a double-resistant LB solid medium containing kanamycin and chloramphenicol. Picking with a monomida Follow 5 ml of kanamycin containing 50 μg / ml of lt liquid medium, at 37 ° C, 200 rpm shock and cultured overnight. Press 1: 100 ratio to the 500 ml shake flask containing 200 ml of medium; the growth curve of E. coli is measured with an UV spectrophotometer, and the concentration of the bacterial liquid is OD after E. coli. 600 At 0.8 mm, E. coli was induced by 0.8 mM IPTG, and E. coli was collected after 12 h, 250 rpm.

[0047] 2 separation and purification of fusion protein MP-AFP

[0048] Escherichia coli fermentation broth was centrifuged at 4 ° C, 6000 rpm. The primary bacterium is resuspended with a PBS solution, the bacterial liquid is ultrasound under the ice water b...

Embodiment 3

[0054] Example 3: Surface modification and atomic force microscopy analysis

[0055] 1 Sample surface modification of the matrix material

[0056] The glass sheet and the mica were matrix material, the surface of the sample modified matrix material with water and fusion protein MP-AFP. The cleansing of the cleansing matrix material was infiltrated in two samples at 25 ° C for 12 h at 25 ° C, and at least 3 repetitions per group were incubated at 25 ° C. After the incubation, the matrix material was washed 3 times with high purity water to remove excess protein samples.

[0057] 2 atomic force microscope detection

[0058] The surface morphology of the matrix material is detected by the atomic force microscope, and the surface of the fusion protein modified glass sheet is compactly dense protein trimming layer, and the regular order.

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Abstract

The present invention relates to a diblock fusion protein with dual functions of adhesion-antifreeze and its synthesis method and application; a gene expression carrier containing antifreeze protein gene and adhesion protein gene is prepared by using genetic engineering as a technical means, And transform it into host cells, make it overexpress the protein by inducer, separate and purify to obtain the fusion protein. The amino acid sequence of the fusion protein is SEQ ID NO.3, and the nucleotide sequence is SEQ ID NO.4. Mussel adhesion protein and antifreeze protein were fused at the gene level to construct a two-block fusion protein with dual functions of adhesion and antifreeze; the fusion protein was expressed by Escherichia coli fermentation, and purified by affinity chromatography Albumin, using acetic acid to purify inclusion bodies, constructs a high-purity separation and purification method, and realizes the one-step synthesis of fusion proteins by microorganisms. This process is mild and efficient, low in cost, low in equipment requirements, and easy to expand production in the later stage.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and more particularly to two-block fusion proteins and synthesis methods and applications having adhesive-antifreeze double function; including gene design of two-block fusion proteins having adhesion-antifreeze dual function, Construction of recombinant expression vectors containing a fusion protein gene fragment, fermentation expression, fusion protein separation, and its application in antifreeze surfaces in E. coli. Background technique [0002] The antifreeze protein (AFP) is a class of protein compounds having improved biological antifreeze resistance, which can bind to small crystals, prevent ice crystallization and crystal growth. Typically, the larger the resection of the antifreeze protein is, the better the organism to low temperature. (The thermal hysteresis effect is a refrigeration of the solution in a non-agreed, and does not change its melting point, so that the ice point and melti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N15/65
CPCC07K14/43504C07K14/43563C12N15/70C12N15/65C07K2319/00
Inventor 张雷杨静张相宇高弈航
Owner TIANJIN UNIV
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