CN116693692B - Recombinant mussel mucin and preparation method thereof - Google Patents
Recombinant mussel mucin and preparation method thereof Download PDFInfo
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- CN116693692B CN116693692B CN202310372127.5A CN202310372127A CN116693692B CN 116693692 B CN116693692 B CN 116693692B CN 202310372127 A CN202310372127 A CN 202310372127A CN 116693692 B CN116693692 B CN 116693692B
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- 241000237536 Mytilus edulis Species 0.000 title claims abstract description 38
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- AHMIDUVKSGCHAU-LURJTMIESA-N L-dopaquinone Chemical compound [O-]C(=O)[C@@H]([NH3+])CC1=CC(=O)C(=O)C=C1 AHMIDUVKSGCHAU-LURJTMIESA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K6/00—Preparations for dentistry
- A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Oral & Maxillofacial Surgery (AREA)
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Abstract
The invention discloses recombinant mussel mucin and a preparation method thereof. The amino acid sequence structure of rMfp151,151 protein is as follows: the mature Mcfp-5 peptide derived from Mytilus coruscus Mytilus coruscus is fused at both ends to the natural peptide fragment of Mytilus coruscus Mytilus edulis Mefp-1, and a hexahistidine tag is added at the C-terminus. According to the invention, three genes rMfp, TYR and ORF438 are constructed into a double-plasmid expression system, so that the high expression of the hydroxylation modified co-rMfp151 protein is realized. The purification method can obtain the target protein with the purity of more than 90 percent, and can be solidified at room temperature to finish the adhesion of the adhered object. The problems of low extraction rate, high cost, low expression level of recombinant mussel mucin, difficult purification and poor viscosity caused by low in-vitro hydroxylation rate of natural mussel mucin are solved.
Description
Technical Field
The invention relates to mussel mucin, in particular to recombinant mussel mucin and a preparation method thereof.
Background
Marine mussels belong to the phylum mollusca, class of the lamellipoda, most of which belong to the mussel family (MYTILIDAE), and are a ubiquitous species of organisms both coastal and offshore. The foot silk gland can secrete foot silk and form an adhesion disc at the tail end of the foot silk to be attached to the substrate, so that mussels can still be tightly attached to the substrate under the flushing of billows. The main component of the adhesive disk is mussel mucin (Mussel Adhesive Protein, MAP; also called mussel podoglobin Mussel footprotein, mfp) which has high strength, high toughness and water resistance, and the viscosity of the adhesive disk exceeds most of mucilage glue, so that the adhesive disk has wide application prospect: the adhesive can be used for bonding broken bones, repairing tooth cracks, bonding soft tissues, repairing skin wounds and the like in clinical aspects; can also be used as an adsorption medium for cell culture; the method has potential application value in the aspects of underwater operations such as water prevention, antibacterial and sealing of seawater equipment.
MAPs are positively charged basic proteins with isoelectric Points (PI) between 8 and 11, contain high abundance of 3, 4-dihydroxyphenylalanine (Dopa ), and are hydroxylated forms of tyrosine residues. Dopa is used as an o-hydroxy phenol derivative and can be bonded with the surfaces of various substrates through non-covalent bonds and covalent bonds to form strong adhesive force. Meanwhile, the oxidation-reduction state of dopa has a great influence on MAP adhesion performance, and the reduced dopa has a strong bonding force with inorganic surfaces. The design of cured in place materials and adhesion of biological substrates requires dopaquinone in an oxidized state.
To date, 6 types of mussel mucins, MAP-1 to MAP-6 proteins, have been identified that are associated with an adhesive function, which exhibit localization of the function and perform different functions. Among them, MAP-1, MAP-3 and MAP-5 are main adhesion functional molecules. MAP-1 is the most recently discovered and studied mussel mucin, whose molecular structure has a similar ten skin repeat, in which dopa is the structural basis for adhesive activity. MAP-3 has the smallest molecular weight among the six adhesion proteins, but its dopa content is high, so it can be directly attached to the surface of the medium, resulting in strong surface interactions. MAP-5 is the highest in dopa content in all mussel mucins, and also has higher positive charge, and is one of key proteins of mussel foot-filament viscose interfaces.
At present, mussel mucin is still mainly obtained by extraction, the yield and the efficiency are extremely low, and the problems that the expression level of natural MAP-1, MAP-3 and MAP-5 sequences are low, the purification yield is low and the industrialization is difficult to realize exist, such as （【1】Silverman H G , FF Roberto. Cloning and expression of recombinant adhesive protein Mefp-1 of the blue mussel, Mytilus edulis: US,US6987170 B1[P]. 2006. 【2】Dong S H , Gim Y , Cha H J . Expression of FunctionalRecombinant Mussel Adhesive Protein Type 3A in Escherichia coli[J]. BiotechnolProg, 2010, 21(3). 【3】Hwang DS, Yoo HJ, Jun JH, Moon WK, Cha HJ. Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli.ApplEnviron Microbiol. 2004;70:3352–3359. 【4】 Lv Yuwei, lv Yawei, yang Zeshang, wang Rui, zhang Yujing and Wang Yingjuan, the prokaryotic expression and the purification of mussel protein Mgfp-5 are [ J ]. University of northwest (natural science edition), 2016,46 (03): 390-396 [5 ] Lv Yawei, wang Rui, zhang Yujing, gao Wenying, yang Ze and Wang Yingjuan, the expression and the functional evaluation of recombinant mussel mucin Mgfp-5 are [ J ]. Genomics and applied biology, 2017,36 (10): 4108-4115 ].
Disclosure of Invention
The invention aims to solve the problems of low extraction rate, high cost, low expression level, difficult purification, low in-vitro hydroxylation rate and poor viscosity of natural mussel mucin, and provides a preparation method of the recombinant mussel mucin with high expression level, high hydroxylation rate, simpler purification, high viscosity and the like.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a preparation method of recombinant mussel mucin comprises the following steps:
(1) Preparing BL21 (DE 3)/rMfp 151 competent cells;
(2) rMfp151 co-expression of Tyrosinase (TYR) and its cofactor (ORF 438) (co-expressed rMfp protein hereinafter abbreviated as co-rMfp151 protein);
(3) Purifying the co-rMfp protein and carrying out hydroxylation identification on tyrosine in the co-rMfp protein;
compared with the prior art, the invention has the following beneficial effects:
(1) Based on the problems of the existing natural mussel mucin extraction and recombinant mussel mucin expression purification, the invention designs from the aspects of genes and protein sequences, and comprises a Mytilus edulis Mefp-1 peptide segment and Mytilus coruscus Mcfp, wherein the peptide segment comprises high-abundance tyrosine and has high structural stability. In addition, the hexahistidine tag is added at the C terminal, so that subsequent identification and purification are facilitated, and the mussel mucin novel molecule rMfp151,151 is finally obtained. Stability predictions for fp151 and rMfp151 with ProtParam Tool showed that rMfp151 had a significantly lower instability coefficient of 37.18 compared to 44.19 (> 40 unstable, <40 stable) of fp151, a structurally stable molecule.
(2) In expression, the present invention employs an intracellular modification scheme for co-expression of Tyrosinase (TYR) and its cofactor (ORF 437). And optimizing three genes rMfp, TYR and ORF438 according to E.coli codon preference, selecting pACYCDuet double expression frame plasmid to express Tyrosinase (TYR) and auxiliary factor (ORF 438) thereof, expressing rMfp151 protein by pET20b, and finally constructing a double plasmid expression system to realize high expression of co-rMfp151 (co-expression rMfp 151) in BL21 (DE 3). The scale induction of 500mL shake flask (common conical flask) is carried out for 3.5h, the expression quantity of co-rMfp protein can reach 37.5% of the total protein of the thallus, the yield of the target protein after purification of Ni affinity chromatography tandem G25 is 70-80mg/L, and the HPLC purity reaches 94.69%. Culturing in a fermentation tank 5L, and purifying the co-rMfp151 protein to obtain the product with the yield of 250-300mg/L.
(3) The invention provides a complete recombinant expression rMfp-151 protein process, from construction, fermentation, purification and identification of engineering escherichia coli, and specific operation and parameters of each step are disclosed in detail, and the process is stable. The recombinant co-rMfp-151 protein is purified by adopting Ni affinity chromatography tandem G25, and finally the target protein with the purity of more than 90 percent can be obtained. The concentrated solution of the co-rMfp-151 protein can be solidified at room temperature to finish the adhesion of the adhered objects.
Drawings
FIG. 1 is a rMfp expression condition optimization diagram;
1. Not induced; m, protein molecular weight standard (14.4-116 kD, non-prestaining); 2. induction with 1.0Mm IPTG at 37℃for 3.5h; 3. induction with 0.5Mm IPTG at 37℃for 3.5h; 4. induction with 0.25Mm IPTG at 37℃for 3.5h; 5. induction with 0.1Mm IPTG at 37℃for 3.5h; 6. induction with 1.0Mm IPTG at 30℃for 6h; 7. inducing with 0.5Mm IPTG at 30 ℃ for 6h; 8. induction with 0.25Mm IPTG at 30℃for 6h; 9. inducing with 0.1Mm IPTG at 30 ℃ for 6h; 10. induction with 1.0Mm IPTG at 20℃overnight; 11. induction with 0.5Mm IPTG at 20 ℃ overnight; 12. induction with 0.25Mm IPTG at 20 ℃ overnight; 13. induced overnight at 20℃with 0.1mM IPTG.
FIG. 2 is a graph of gray scale analysis of rMfp for expressing the condition optimization results;
1. not induced; m, protein molecular weight standard (14.4-116 kD, non-prestaining); 2. induction with 1.0Mm IPTG at 37℃for 3.5h; 3. induction with 0.5Mm IPTG at 37℃for 3.5h; 4. induction with 0.25Mm IPTG at 37℃for 3.5h; 5. induction with 0.1Mm IPTG at 37℃for 3.5h; 6. induction with 1.0Mm IPTG at 30℃for 6h; 7. inducing with 0.5Mm IPTG at 30 ℃ for 6h; 8. induction with 0.25Mm IPTG at 30℃for 6h; 9. induction with 0.1Mm IPTG at 30℃for 6h
FIG. 3 is a graph of optimization of co-rMfp expression conditions;
1. Not induced; m, protein molecular weight standard (14.4-116 kD, non-prestaining); 2. induction with 1.0Mm IPTG at 37℃for 3.5h; 3. induction with 0.5Mm IPTG at 37℃for 3.5h; 4. induction with 0.25Mm IPTG at 37℃for 3.5h; 5. induction with 0.1Mm IPTG at 37℃for 3.5h; 6. induction with 1.0Mm IPTG at 30℃for 6h; 7. inducing with 0.5Mm IPTG at 30 ℃ for 6h; 8. induction with 0.25Mm IPTG at 30℃for 6h; 9. inducing with 0.1Mm IPTG at 30 ℃ for 6h; 10. induction with 1.0Mm IPTG at 20℃overnight; 11. induction with 0.5Mm IPTG at 20 ℃ overnight; 12. induction with 0.25Mm IPTG at 20 ℃ overnight; 13. induction was carried out overnight at 20℃with 0.1mM IPTG (co-rMfp 151, tyrosinase and tyrosinase cofactor ORF438 in the order indicated by the arrow from top to bottom).
FIG. 4 is a gray scale analysis chart of the results of optimization of co-rMfp expression conditions;
M, protein molecular weight standard (14.4-116 kD, non-prestaining); 1. Induction with 1.0Mm IPTG at 37℃for 3.5h; 2. induction with 0.5Mm IPTG at 37℃for 3.5h; 3. Induction with 0.25Mm IPTG at 37℃for 3.5h; 4. IPTG induction at 37℃for 3.5h at 0.1 mM.
FIG. 5 is a purification diagram of co-rMfp;
1. A supernatant; 2. precipitating; 3. flow through; 4. washing impurities; 5. eluting; m, protein molecular weight standard (14.4-116 kD, non-prestaining).
FIG. 6 is an HPLC purity profile of co-rMfp;
FIG. 7 is a diagram of a hydroxyl assay for rMfp and co-rMfp 151;
FIG. 8 is a graph showing adhesion of co-rMfp protein to pp and PS materials;
FIG. 9 is a LC-MS total ion flow chromatogram of a co-rMfp protein sample.
Detailed Description
Example 1, rMfp recombinant expression
The method comprises the following steps:
S1, cloning: optimizing a protein sequence rMfp according to the codon preference of escherichia coli, adding a 6xHis-tag sequence at the C end, and enabling the complete gene sequence to be shown as SEQ ID NO. 1; and cloning the gene sequence into an expression vector pET20b to obtain an expression vector containing rMfp protein sequences. The pET20b-rMfp plasmid was transformed into BL21 (DE 3) by freeze thawing, and screened on LB-Amp plates. Single colonies were picked and bacterial liquid PCR was performed.
The reaction system: 1. Mu.L of the bacterial liquid, rMfp-F/R0.5. Mu.L, 1.1xT3 mix (Optimum Praeparata) 18. Mu.L.
Primer sequence: rMfp151-F:5'-CATATGGCTATCATGAATCACAAAGAG-3' (5'-CATATGGCTATCATGAATCACAAAGAG-3'),
RMfp151-R:5'-CTTAGTGATGGTGATGATGGTGCTTATAG-3'. Reaction conditions: 98℃2min,98℃10s,59℃10s,72℃15s,72℃3min (30 cycles). After the reaction, 1% agarose gel electrophoresis was performed, and ultraviolet inspection was performed.
S2, screening of high-expression strains
The positive monoclonal is inoculated into 5mL LB-Amp culture medium according to a proportion of one thousandth, cultured overnight at 37 ℃ at 200rpm, then transferred into 5mL LB-Amp culture medium according to five thousandths, cultured for about 3 hours at 220rpm at 37 ℃ until the OD600 is 0.6-0.8, and added with 1mM IPTG with the final concentration for induction expression. Inducing at 37 ℃ at 200rpm for 3.5 hours, collecting thalli at 12000rpm, and keeping at-20 ℃ for later use.
The whole fungus sample is cracked by 50 mu L of 5% SDS, 50 mu L of 2xLoadingBuffer is added, boiling water bath is carried out for 10min, centrifugation is carried out at 12000rpm, 8 mu L of supernatant is sampled (12% separating gel and 5% concentrating gel), low pressure is carried out for 30min, high pressure is carried out for 120v for 66min, electrophoresis is carried out, and examination is carried out. Screening out the strain with the highest expression amount and freezing.
S3, expression condition optimization
The glycerinum is inoculated into 5mL LB-Amp culture medium according to a thousandth proportion, cultured overnight at 37 ℃ at 200rpm, transferred into 5mL LB-Amp culture medium according to five thousandths, and added with final concentration of 1mM, 0.5mM, 0.25 mM and 0.1 mM IPTG for induction expression when the OD600 is 0.6-0.8 after culturing at 37 ℃ at 220 rpm. The cells were induced at 37℃and 30℃and 20℃at 200rpm for 3.5 hours, 6 hours and overnight, respectively, and SDS-PAGE was performed on the cells, and the results are shown in FIG. 1 and FIG. 2. The highest expression level at 37 ℃ and the expression level of the target protein at the IPTG concentration of 0.5mM are 40.5 percent of the total protein of the bacterial body.
Example 2 recombinant expression of co-rMfp151
The method comprises the following specific steps:
S1, cloning: optimizing protein sequences TYR and ORF438 according to the preference of escherichia coli codons, wherein the complete gene sequences are shown as SEQ ID NO.2 and SEQ ID NO. 3; the gene sequence is cloned into an expression vector pACYCDuet to obtain an expression vector pACYCDuet-TYR-ORF438 containing TYR and ORF438 protein sequences.
The pACYCDuet-TYR-ORF438 plasmid was transformed into high expression engineering bacterium BL21 (DE 3)/rMfp 151 competent (prepared by CaCl 2 method) by freeze thawing method, and LB-Cm-Amp plate screening was performed. Single colonies were picked for bacterial liquid PCR. The reaction system: 1) 1. Mu.L of the bacterial liquid, rMfp-F/R0.5. Mu.L, 1.1xT3 mix (Optimum Praeparata) 18. Mu.L. 2) 1. Mu.L of the bacterial liquid, 0.5. Mu.L of Tyr-F/R, and 18. Mu.L of 1.1xT3 mix (Optimago). Primer sequence: tyr-F:5'-ATGACCGTTCGCAAAAACCAG-3', tyr-R:5'-caTTAAACATCAAAGGTATAATGACGGGTATG-3'. Reaction conditions: 98℃2min,98℃10s,59℃10s,72℃15s,72℃3min (30 cycles). And (5) carrying out nucleic acid electrophoresis and ultraviolet inspection after the reaction is finished.
S2, screening of high-expression strains
The strong positive monoclonal is inoculated into 5mL LB-Amp culture medium, cultured overnight at 37 ℃ at 200rpm, transferred into 5mL LB-Amp culture medium by five thousandths, and added with IPTG with the final concentration of 1mM when the OD600 is 0.6-0.8 after culturing at 220rpm at 37 ℃ for induction expression. Inducing at 37 deg.c and 200rpm for 3.5 hr, collecting thallus, SDS-PAGE electrophoresis and sieving out the strain with highest expression level.
S3, expression condition optimization
The high-expression bacteria are inoculated into 5mL LB-Cm-Amp culture medium according to the proportion of one thousandth, cultured overnight at 37 ℃, transferred into 5mL LB-Cm-Amp culture medium according to five thousandths, and added with final concentration of 1mM, 0.5 mM, 0.25mM and 0.1 mM IPTG for induction expression when the OD600 is 0.6-0.8 after culturing at 37 ℃ at 220 rpm. Inducing at 37deg.C, 30deg.C and 20deg.C at 200rpm for 3.5 hr, 6 hr and overnight respectively, taking whole bacteria sample, and performing SDS-PAGE electrophoresis to obtain the results shown in FIG. 3 and FIG. 4. The target protein expression level is relatively highest at 37 ℃ and 0.25mM IPTG, and reaches 37.5% of the total protein of the bacterial cells.
Example 3, co-rMfp151 500mL Scale shake flask, purification and purity detection
S1, co-rMfp purification
1. Preparing a solution:
Denaturation buffer: 8M urea, 0.5M NaCl, 20mM PB (pH 8-7.5),
Renaturation buffer: 4M urea, 0.5M NaCl, 20mM PB (pH 8-7.5),
Washing impurity buffer solution: 500mM imidazole, 0.5M NaCl, 20mM PB (pH 6.6-pH 6.0),
Elution buffer: 600mM imidazole, 0.5M NaCl, 20mM PB (pH 5.0-pH 2.0);
2. and (3) purification:
3 column volumes of the Ni column are balanced by using a denaturation buffer solution, the Ni column is suspended according to the ratio of the thallus to the denaturation buffer solution=1:5, the temperature is 4 ℃, the pressure is high and homogenized for 3 times at 800ba, the centrifugation is carried out for 20min at 15000rpm, the supernatant is collected and loaded, the flow-through liquid is collected in the process, and the Ni column is balanced by using the denaturation buffer solution for 5-8 column volumes after the loading is finished. Renaturation is carried out by using renaturation buffer solution, and the column is washed for 3 to 5 column volumes. And (5) replacing the impurity washing buffer solution for washing 3-5 column volumes, replacing the eluting buffer solution for eluting the target protein after the base line is stable, and collecting eluting peaks, wherein the result is shown in figure 5.
HPLC purity determination of S2, co-rMfp151
1. Separation column information:
5μm C18（2）100Å
LC Column 250×4.6 mm
2. Mobile phase:
flow rate: 0.8 mL/min
Phase A water
Phase B methanol
Phase C0.1% TFA-acetonitrile
Phase D0.1% TFA-water
3. Time: 35 min
Time program:
0-30min C.Conc 70% D.Conc 30%；
30-35min C.Conc 5% D.Conc 95%
4. Automatic sample injector:
Sample suction speed: 5.0 Mu L/sec
Sample injection amount: 80 mu L
Sample cooler temperature: 4. DEG C
5. Wavelength:
wavelength acquisition range: 190 nm-800 nm
6. Results: see fig. 6.
Example 4, hydroxylation verification of co-rMfp151, MS identification and hydroxylation Rate determination
S1, hydroxy assay
1. Preparing a solution:
(1) 2M potassium glycinate solution: 46.9g glycine is dissolved in 200mL purified water, the pH value is adjusted to 10.0 by KOH, the volume is fixed to 250mL, and the glycine is preserved at 4 ℃ for standby;
(2) 0.16M sodium borate solution: 15.6g sodium borate is dissolved in 200mL purified water, and the volume is fixed to 250mL, and the mixture is preserved at normal temperature for standby.
2. Spotting: a0.22 μm aqueous film was taken and marked with a pencil at the spot. Taking protein sample solution (rMfp sample solution, co-rMfp protein sample solution and solvent sample) 2 mu L each time, applying the sample solution to the marked place, and continuing to apply sample for the next time after the sample solution is dried, and repeating for a plurality of times until the application amount reaches about 6 mu g of protein (the application amount of the solvent group is the same as the application volume of the protein sample solution).
3. Pretreatment: after the water had volatilized, the membrane was placed in a beaker, 300mL of purified water was added, and sonicated for 10min.
4. Dyeing: the film was transferred to a suitable container, added with NBT-potassium glycinate staining solution, and tinfoil wrapped for 45min in dark.
5. Washing and developing: the NBT-potassium glycinate staining solution was discarded, washed 2 times with sodium borate solution and left overnight in sodium borate solution. The membrane was rinsed with purified water, and the membrane was stored in purified water, and the stored result was photographed in time, see fig. 7. The co-rMfp 151,151 samples showed a pronounced mauve at the spots, the mauve of the non-hydroxylated samples was hardly visible, and no spots were found at the spots of the solvent set.
S2, adhesion test
1. Sample preparation: placing the purified co-rMfp151 into a dialysis bag with the activated cutoff molecular weight of 10kD, dialyzing with 5% acetic acid aqueous solution, changing the dialyzate every 12h, transferring the sample into a proper container after the last dialysis, and performing freeze drying after gradient cooling.
2. Adhesion experiments: 0.5-1mg of the freeze-dried co-rMfp protein is taken and dissolved in2 mu L of 5% acetic acid aqueous solution, the dissolved protein solution is coated on the base part (the suction head material is Polypropylene (PP)) of a suction head of 10 mu L and 200 mu L, and then the suction head is placed on a culture dish made of polystyrene (Polystyrene, abbreviated PS) and cured for about 3 hours at room temperature. After solidification, it was observed that the tips adhered to the surface of the dish, and the inverted dish tips remained in the dish-adhered state, see FIG. 8.
S3, mass spectrum identification and hydroxylation rate determination
1. Sample pretreatment
(1) SDS-PAGE gel cutting purification
(2) Enzymatic hydrolysis of pancreatic protein (Trypsin)
2. LC-MS/MS detection
The co-rMfp protein sample is subjected to LC-MS/MS data acquisition to generate a raw file, and the raw file is subjected to software Byonic database retrieval to obtain an identification result, wherein the result shows that the protein coverage is 98.3%; 22 tyrosine (Y) hydroxylation sites were detected, accounting for 38.6% of all Y's of the protein, accounting for 7.48% of the total amino acids of the protein. The LC-MS total ion flow chromatogram is shown in FIG. 9.
Claims (10)
1. The recombinant mussel mucin is characterized in that the two ends of Mcfp-5 mature peptide derived from Mytilus coruscus Mytilus coruscus are fused with a Mefp-1 peptide fragment of Mytilus edulis to obtain a new mussel mucin molecule rMfp151, and the amino acid sequence of the new mussel mucin molecule is shown as SEQ ID NO. 1.
2. The recombinant mussel mucin of claim 1, further comprising a His tag for protein purification.
3. Nucleic acid encoding a recombinant mussel mucin according to claim 1 or 2, and subjected to e.coli preferential codon optimisation with the sequence SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4.
4. The nucleic acid according to claim 1, having the sequence shown in SEQ ID NO. 4.
5. The nucleic acid of claim 3, further comprising a His tag for protein purification.
6. A vector for expressing the recombinant mussel mucin, which is characterized by being pET28a, pET20b or pQE30.
7. A host cell comprising the expression vector of claim 6, characterized by being BL21-DE3, origami-DE3 or M15.
8. A method for preparing the recombinant mussel mucin according to claim 1 or 2, comprising the steps of:
(1) Preparation of rMfp competent cells: optimizing a protein sequence rMfp according to the codon preference of escherichia coli, cloning the obtained gene sequence into an expression vector, transforming pET20b-rMfp151 into competent cells of an expression strain by adopting a freeze thawing method, and preparing rMfp151 competent cells of the high expression strain obtained by screening by adopting a CaCl 2 method;
(2) Expression of co-rMfp151 recombinant protein: optimizing protein sequences TYR and ORF438 according to the preference of E.coli codons to obtain gene sequences shown as SEQ ID NO.6 and SEQ ID NO. 7; cloning the two gene sequences into an expression vector pACYCDuet to obtain an expression vector pACYCDuet-TYR-ORF438 containing TYR and ORF438 protein sequences; transforming pACYCDuet-TYR-ORF438 plasmid into BL21-DE3/rMfp151 competent cells obtained in the step (1), and screening to obtain co-rMfp151 high-expression strain;
(3) Purification of co-rMfp protein: and purifying by adopting Ni affinity chromatography and tandem G25 to obtain the high-purity co-rMfp-151 recombinant protein.
9. The method for preparing recombinant mussel mucin according to claim 8, wherein the purification of co-rMfp protein in step (3) comprises eluting the protein under conditions of 500mM imidazole, 0.5M NaCl, 20mM PB, pH7.0-pH6.0 in a wash buffer, and eluting the protein of interest under conditions of 600mM imidazole, 0.5M NaCl, 20mM PB, pH5.0-pH1.0 in a wash buffer.
10. Use of the recombinant mussel mucin according to claim 1 or 2 for the preparation of a bioadhesive or cell culture adsorption medium for the bonding of fractured bones, the repair of dental fissures, the bonding of soft tissues, the repair of skin wounds.
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| CN115819627A (en) * | 2022-12-28 | 2023-03-21 | 西安德诺海思医疗科技有限公司 | Bionic recombinant type III mussel mucin as well as preparation method and application thereof |
| CN115894655A (en) * | 2022-12-28 | 2023-04-04 | 西安德诺海思医疗科技有限公司 | Bionic recombinant V-type mussel mucin as well as preparation method and application thereof |
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| CN115819627A (en) * | 2022-12-28 | 2023-03-21 | 西安德诺海思医疗科技有限公司 | Bionic recombinant type III mussel mucin as well as preparation method and application thereof |
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