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CN-122011149-A - Type VI recombinant mussel foot protein and preparation method and application thereof

CN122011149ACN 122011149 ACN122011149 ACN 122011149ACN-122011149-A

Abstract

The invention discloses a VI type recombinant mussel foot protein, a preparation method and application thereof, wherein the amino acid sequence of the VI type recombinant mussel foot protein is shown as SEQ ID NO. 4. The recombinant mussel foot protein VI can be used as a modifier for improving the hydroxylation efficiency of mussel mucin, and can improve the hydroxylation efficiency of mussel mucin to 40-45%, so that mussel mucin with high dopa content is obtained. The recombinant type VI mussel foot protein has high reducing power and good biocompatibility, has no toxicity to cells, and can be used as biomedical materials and cosmetic raw materials.

Inventors

  • MA YI

Assignees

  • 华南理工大学

Dates

Publication Date
20260512
Application Date
20260119

Claims (10)

  1. 1. The recombinant mussel foot protein VI is characterized in that the amino acid sequence of the recombinant mussel foot protein VI is shown as SEQ ID NO. 4.
  2. 2. A gene sequence encoding the recombinant mussel foot protein of claim 1, wherein the gene sequence is shown in SEQ ID No. 2.
  3. 3. The preparation method of the VI-type recombinant mussel foot protein is characterized by comprising the following steps: (1) Connecting a gene sequence encoding the recombinant mussel foot protein of claim 1 to a vector plasmid, and introducing the synthesized plasmid into engineering bacteria; (2) Inoculating the engineering bacteria into an LB liquid culture medium, fermenting and culturing until OD600 = 0.6-0.8, performing fermentation for 6-8 hours after induction, and collecting thalli after finishing fermentation; (3) BufferA after re-suspending the collected thalli, carrying out ultrasonic crushing, centrifugally collecting supernatant, adsorbing recombinant protein by a nickel column, and then using a BufferB gradient containing different imidazole concentrations to elute the hybrid protein and recovering the recombinant protein; (4) And (5) dialyzing and freeze-drying the recovered recombinant protein to obtain the recombinant mussel foot protein VI.
  4. 4. The method according to claim 3, wherein the gene sequence in step (1) is shown in SEQ ID NO. 2.
  5. 5. The method of claim 3, wherein the carrier plasmid in the step (1) is pET28a, the engineering bacterium is E.coli BL21 (DE 3), and the fermentation condition in the step (2) is 37+ -2 ℃ and the rotation speed is 220+ -50 rpm.
  6. 6. Use of a recombinant mussel foot protein of type VI according to claim 1 or a recombinant mussel foot protein of type VI prepared by the method of any one of claims 3-5 as a modifier for increasing the efficiency of hydroxylation of mussel mucin.
  7. 7. The use according to claim 6, characterized in that it comprises the steps of: (1) Dissolving type VI recombinant mussel foot protein, and adding mussel mucin; (2) Tyrosinase was added to carry out catalytic reaction.
  8. 8. The use according to claim 7, wherein the mass ratio of mussel mucin to type VI recombinant mussel foot protein in step (1) is 9±3:1.
  9. 9. The use according to claim 7, wherein the tyrosinase in step (2) is agaricus bisporus tyrosinase, the tyrosinase is added in an amount of 1.0 ± 0.5wt.% of mussel mucin, and the mussel mucin is a tyrosine-rich mussel mucin.
  10. 10. The use according to claim 7, 8 or 9, wherein the recombinant mussel foot protein type VI in step (1) is dissolved in PBS buffer, and the reaction conditions in step (2) are temperature 25±5 ℃, rotation speed 220±50rpm, ph=6.5±0.5, and reaction time 90±30min.

Description

Type VI recombinant mussel foot protein and preparation method and application thereof Technical Field The invention relates to the field of recombinant proteins and biocatalysis, in particular to construction, expression and purification of recombinant mussel mucin capable of improving in-vitro hydroxylation efficiency of mussel mucin and application thereof. Background Mussels are able to attach themselves to solid surfaces in marine environments of high salinity, humid, while constantly being attacked by ocean waves, because the podites, which directly interact with the surface, produce a variety of mussel podophylloids (Mussel foot protein, MFP), which interact to exert an adhesive effect, and therefore this protein is also known as mussel mucin (Mussel adhesive protein, MAP). The current studies indicate that type III and type V proteins that are rich in tyrosine residues are mainly responsible for adhesion and that these tyrosine residues can be converted by hydroxylation to the atypical amino acid 3, 4-dihydroxyphenyl-L-alanine (Dopa ). Dopa can participate in a variety of non-covalent interactions, such as hydrogen bonding, cation-pi interactions, pi-pi interactions. In addition, dopa can also participate in various chemical interactions, such as formation of metal-dopa complexes by coordination chemistry. These physical and chemical interactions are involved in the underwater adhesion of dopa. In addition to adhesion, the high reducibility of dopa provides antioxidant function to proteins, and mussel mucins have shown potential as cosmetic raw materials and medical devices. Because the cost of the naturally extracted mussel mucin is extremely high, the cost can be reduced by using the microorganism to express the recombinant mussel mucin exogenously, and the content of the functional protein is greatly improved. However, the in vitro hydroxylation of dopa is extremely easy to be excessively catalyzed, so that oxidative cross-linked dopa quinone is generated, and the functions of recombinant mussel mucin are greatly weakened. The modifier in the existing hydroxylation system is mainly sodium borate and vitamin C, wherein the sodium borate has the function of dispersing and exposing hydroxylation sites of proteins, and the vitamin C is used for protecting dopa from being excessively oxidized. Although the in vitro hydroxylation efficiency of the mussel mucin of the hydroxylation system is 20-25%, compared with the hydroxylation system without the modifier, the hydroxylation efficiency is improved, the mussel mucin needs to be subjected to secondary dialysis and recovery after hydroxylation due to the addition of other reagents, so that the loss is increased, and the pre-application treatment of the recombinant mussel mucin is more complicated. Therefore, how to efficiently increase the hydroxylation rate of the in vitro hydroxylation mussel mucin is an important research direction in the future. Disclosure of Invention The invention aims to provide a preparation method and application of recombinant VI mussel foot protein, optimize a hydroxylation reaction system for hydroxylating tyrosine residues in recombinant mussel mucin into dopa, realize the improvement of the efficiency of hydroxylation outside a tyrosine residue matrix, and simultaneously protect the dopa from being excessively oxidized into dopa quinone to lose the function of the dopa. The recombinant mussel foot protein of the VI type is successfully expressed and purified by screening through NCBI, constructing a mussel foot protein gene expression plasmid of the VI type and transferring the mussel foot protein gene expression plasmid into engineering strains to realize expression, and the in vitro hydroxylation efficiency is improved from 20-25% to 40-45% by mixing and catalyzing the purified recombinant mussel foot protein of the VI type and the V-type or III-type mussel mucin and tyrosinase. The invention is carried out according to the following scheme for achieving the above aim: The invention is designed based on the known natural VI mussel foot protein sequence. And mixing the successfully expressed and purified type VI recombinant protein with type III and type V mussel mucin rich in tyrosine residues, and carrying out hydroxylation modification on the mixture by tyrosinase, thereby obtaining the mussel mucin with high dopa content. The mussel foot protein sequence of type VI is rich in the gene sequence of cysteine residues and glycine residues, wherein the cysteine residues provide reducing power, and the glycine residues can be rich in peptide self-assembly. A recombinant mussel foot protein of type VI has an amino acid sequence shown in SEQ ID NO. 4. The gene sequence of the VI type recombinant mussel foot protein is shown as SEQ ID NO. 2. A preparation method of VI type recombinant mussel foot protein comprises the following steps: (1) Connecting a gene sequence encoding the recombinant mussel foot protein of claim 1 to a ve