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Mussel attachment protein/amphoteric ion polypeptide fusion protein with attachment-anti-staining dual functions and synthesis method

A technology of mussel adhesion protein and fusion protein, which is applied in the directions of fusion polypeptides, chemical instruments and methods, antibody mimics/scaffolds, etc., can solve the problems of high cost and low output, and achieves low cost, low equipment requirements, Anti-fouling effect is obvious

Inactive Publication Date: 2018-09-07
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mussel adhesion protein was originally extracted directly from mussel foot silk, but there are limitations of high cost and low yield. The development of gene recombination technology has realized the recombinant exogenous expression of mussel adhesion protein by using microorganisms

Method used

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  • Mussel attachment protein/amphoteric ion polypeptide fusion protein with attachment-anti-staining dual functions and synthesis method
  • Mussel attachment protein/amphoteric ion polypeptide fusion protein with attachment-anti-staining dual functions and synthesis method
  • Mussel attachment protein/amphoteric ion polypeptide fusion protein with attachment-anti-staining dual functions and synthesis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: Construction of recombinant expression vector and bacterial strain

[0067] ①Construction of recombinant expression vector

[0068] Using 3 GGGGS repeat sequences as connecting polypeptides to connect fusion protein MP and 20 KE repeat sequences of zwitterionic polypeptides to form fusion protein MP-KE, the gene is coded according to the expression preference of Escherichia coli without changing its amino acid sequence sub-optimization. A NdeI restriction site was added at the 5' end of the gene sequence and a HindIII restriction site was added at the 3' end of the gene. The gene sequence was artificially synthesized and cloned into the Escherichia coli expression vector pET-28a to obtain the recombinant expression vector pET-28a-MP-KE, which contained T7 strong promoter, lca lactose operon, kanamycin-resistant Sex tagging sites and hexahistidine tags, such as figure 1 shown.

[0069] ②Chemical transformation of Escherichia coli

[0070] Thaw competen...

Embodiment 2

[0071] Embodiment 2: Expression and purification of fusion protein MP-KE

[0072] ① Fermentative expression of fusion protein MP-KE in Escherichia coli

[0073] 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. with shaking at 200 rpm. Transfer the E. coli seed solution to a 500ml shake flask containing 200ml medium at a ratio of 1:100. Measure the growth curve of Escherichia coli with a UV spectrophotometer. After the E. coli is transferred, when the concentration of the bacterial solution is OD600=0.8, use 0.8mM IPTG to induce E. coli to express the fusion protein MP-KE, at 37°C, under the condition of 250rpm After continuing to cultivate for 12 h, E. coli cells were collected.

[0074] ②Isolation and purific...

Embodiment 3

[0082] Example 3: In vitro hydroxylation and DOPA validation

[0083] ①In vitro hydroxylation reaction

[0084] The 0.1MPBS system containing 1mg / ml fusion protein MP-KE, 250U / ml tyrosinase, 20mM sodium borate, 25mM ascorbic acid was stirred at 25°C and bubbled for 5h to hydroxylate the tyrosine residue in the fusion protein to DOPA . Real-time control of reaction temperature and bubbling speed during the reaction process. After the reaction, the solution was dialyzed, ultrafiltered and freeze-dried.

[0085] ②Nitroblue tetrazolium (NBT) staining

[0086] Three sample solutions of water, fusion protein MP-KE and modified fusion protein were respectively dropped on the polyvinylidene fluoride (PVDF) membrane. After the protein samples were bound to the membrane, soak the PVDF membrane in 2M potassium glycinate (pH 10) solution containing 0.5mg / ml nitroblue tetrazolium (NBT) and treat it in the dark for 1h. The PVDF membrane was washed sequentially with 0.2M sodium borate (...

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Abstract

The invention relates to mussel attachment protein / amphoteric ion polypeptide fusion protein with attachment-anti-staining dual functions and a synthesis method. A fusion protein gene with a rationaldesign is cloned to a plasmid vector to obtain a recombinant expression plasmid; furthermore, after host cells are further transformed, the host cells are induced by utilizing an inducer to excessively express the fusion protein; after separation and purification, the fusion protein is obtained. Mussel attachment protein contains a sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ IDNO. 4 or SEQ ID NO. 5. Amphoteric ion polypeptide contains a sequence shown as SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9. The fusion protein with the attachment-anti-staining dual functions is synthesized through a microorganism one-step method; a process is moderate and efficient, the cost is low and requirements on equipment are low, so that later-period expanded production is facilitated.

Description

technical field [0001] The present invention relates to a mussel adhesion protein / zwitterionic polypeptide fusion protein with dual functions of adhesion and antifouling and a synthesis method thereof, and also relates to the gene design of the fusion protein and the construction of a recombinant expression vector containing fusion protein gene fragments Construction, fermentation expression in Escherichia coli, separation and purification of fusion protein, and application of surface modification to matrix materials. Background technique [0002] Biofouling is a process in which living substances such as bacteria adhere to and multiply on the surface of matrix materials, resulting in equipment loss. In many fields such as medicine, navigation, construction, food, etc., biofouling not only leads to the loss of materials, but also causes problems such as industrial energy consumption and medical safety. For example, when graft materials such as artificial heart valves and vas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70
CPCC07K14/43504C07K2319/00C12N15/62C12N15/70
Inventor 张雷齐海山郑伟伟
Owner TIANJIN UNIV
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