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Diblock fusion protein with adhesion-antifreeze dual functions, and synthesis method and application thereof

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

Active Publication Date: 2020-02-11
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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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: Construction of recombinant expression vector and bacterial strain

[0040] ①Construction of recombinant expression vector

[0041] Three GGGGS repeat sequences were used as the linking polypeptide to connect the adhesion protein and mealworm antifreeze protein to form the fusion protein MP-AFP, and the codon optimization of the gene was carried out according to the expression preference of Escherichia coli without changing its amino acid sequence. A BamHI restriction site was added to the 5' end of the gene sequence and a NotI restriction site was added to the 3' end of the gene; the gene sequence was artificially synthesized and cloned into the E. coli expression vector pET-28a to obtain a recombinant expression vector pET-28a-MP-AFP, the recombinant expression vector contains T7 strong promoter, lca lactose operon, kanamycin resistance marker site and hexahistidine tag, such as figure 1 shown.

[0042] ②Chemical transformation of Escherichia coli

[...

Embodiment 2

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

[0045] ① Fermentative expression of fusion protein MP-AFP in Escherichia coli

[0046] The successfully transformed Escherichia coli was streaked and activated on a double-resistant LB solid medium plate containing kanamycin and chloramphenicol. A single colony was picked and placed in a 5 ml test tube containing 50 μg / ml kanamycin and chloramphenicol LB liquid medium, and cultured overnight at 37° C. and 200 rpm with shaking. Transfer the E. coli seed solution in a 1:100 ratio to a 500ml shake flask containing 200ml medium; measure the growth curve of E. coli with a UV spectrophotometer, and when the concentration of the bacteria solution is OD after E. coli transfer 600 When =0.8, 0.8mM IPTG was used to induce Escherichia coli to express the fusion protein MP-AFP, and culture was continued for 12 hours at 37°C and 250rpm, and then the Escherichia coli cells were collected.

[0047] ②Isolation and p...

Embodiment 3

[0054] Embodiment 3: surface modification and atomic force microscope analysis

[0055] ① Surface modification of the sample on the matrix material

[0056] The glass sheet and the mica sheet are used as matrix materials, and water and fusion protein MP-AFP are used as samples to modify the surface of the matrix material. The cleaned matrix material was soaked in the two samples, and incubated at 25° C. with 80% humidity for 12 hours, with at least 3 repetitions for each group. After incubation, the matrix material was washed 3 times with high-purity water to remove excess protein samples.

[0057] ②Atomic force microscope detection

[0058] The surface morphology of the matrix material was detected by the tapping mode of the atomic force microscope, and the surface of the fusion protein-modified glass slide showed a compact and dense protein-modified layer, which was regular and orderly.

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Abstract

The invention relates to a diblock fusion protein with adhesion-antifreeze dual functions, and a synthesis method and application thereof. By taking genetic engineering as a technological means, a gene expression vector simultaneously containing antifreeze protein genes and adhesion protein genes is prepared and converted into a host cell, through an inducer, the host cell overexpresses the protein, and thus the fusion protein is obtained by separation and purification. The amino acid sequence of the fusion protein is SEQ ID NO.3, and the nucleotide sequence of the fusion protein is SEQ ID NO.4. The mussel adhesion protein and the antifreeze protein are fused on the gene level, and thus the diblock fusion protein with the adhesion-antifreeze dual functions is constructed; and the fusion protein is expressed through fermentation of escherichia coli, supernatant protein is purified through an affinity chromatogram, an inclusion body is purified through acetic acid, a high-purity separation and purification method is constructed, the fusion protein is synthesized through a microbial one-step method, the process is mild and efficient, the cost is low, the equipment requirement degree is low, and expanded production at the later period is easy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a diblock fusion protein with dual functions of adhesion and antifreeze, its synthesis method and application; including the gene design of the diblock fusion protein with dual functions of adhesion and antifreeze, Construction of recombinant expression vector containing fusion protein gene fragment, fermentation expression in Escherichia coli, separation and purification method of fusion protein, and application on antifreeze surface. Background technique [0002] Antifreeze protein (AFP) is a general term for a class of protein compounds that can improve biological antifreeze ability. It can bind to small ice crystals and prevent ice crystallization and crystal growth. Generally, the larger the thermal hysteresis value of the antifreeze protein, the better the adaptability of the organism to low temperature. (The thermal hysteresis effect is to reduce the freezing point...

Claims

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Application Information

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Patent Type & Authority Applications(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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