Mussel mucoprotein synthesis method

Abstract

The invention provides a mussel mucoprotein synthesis method, and relates to the technical field of genetic engineering. The yield of the mussel mucin is increased by performing deletion mutation on an N-terminal sequence of a secondary structure of the mussel mucin with an amino acid sequence such as SEQ ID NO.1, and the yield of the mussel mucin is about 2.57 g / L in the prior art, while the yield of the mussel mucin is about 5g / L, so that the yield of the mussel mucin is greatly increased compared with the yield of the mussel mucin in the prior art. On the basis, the coexpression tyrosinase is used for performing dopa modification on the mussel mucoprotein, and the expression quantity of the tyrosinase is controlled through RBS with different intensities, so that the modification degree of the dopa is regulated and controlled, the yield of the mussel mucoprotein is improved, and meanwhile, controllable modification of the dopa in the mussel mucoprotein is realized; finally, high-yield mussel mucin with different dopa modification degrees is obtained.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a method for synthesizing mussel mucin. Background Art

[0002] Mussel Adhesive Proteins (MAPs) are special proteins evolved by marine mussels to adapt to the rough coastline environment. This protein is mainly found in the byssus of mussels, which can firmly fix mussels on solid surfaces such as rocks and wood, and remain stable even when washed by seawater. The efficient adhesion performance of MAPs has aroused great interest among scientists and has led to application research and development in many fields. This protein contains a large amount of 3,4-dihydroxyphenylalanine (DOPA), which gives it unique strong adhesion, biocompatibility and degradability. Different degrees of DOPA modification in mussel mucin give mussel mucin different functions. Mussel mucin with high DOPA modification can be used in medical bioadhesives, wound repair materials, medical coatings and other fields, while mussel mucin with low DOPA modification is more suitable for skin care products and other fields. Based on these characteristics, mussel mucin and its synthetic analogs show broad application prospects in many fields such as biomedical engineering, paint anticorrosion, and materials science. For example, in biomedicine, it is used to develop new medical adhesives and tissue repair materials; in environmental protection materials, as the basis of marine antifouling coatings, it can reduce pollution and reduce the maintenance costs of ships; in materials science, it has inspired the design of new composite materials, enhancing the materials' moisture resistance, wear resistance and self-healing ability.

[0003] At present, mussel mucin is mainly obtained through tissue extraction. This process includes steps such as raw material preparation, pretreatment, crushing and dissolution, extraction and purification, concentration and drying, and quality control. Various indicators must be strictly monitored throughout the process to ensure the purity, activity and safety of the final product. However, the tissue extraction method faces challenges such as high cost, low yield, uncontrollable DOPA modification and stability issues, which makes it difficult to meet the market demand for mussel mucin. Therefore, a method for synthesizing mussel mucin with high synthesis amount and controllable DOPA modification is urgently needed. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problems in the prior art of low synthesis yield of mussel mucin and uncontrollable degree of DOPA modification.

[0005] In order to solve the above technical problems, the present invention provides a method for synthesizing mussel mucin. The present invention increases the expression amount of mussel mucin by performing deletion mutation on the N-terminal sequence of the secondary structure of the mussel mucin of the amino acid sequence such as SEQ ID NO.1. At the same time, tyrosinase can catalyze the conversion of tyrosine in mussel mucin into ortho-hydroxyl DOPA, so mussel mucin is DOPA-modified by co-expressing tyrosinase, and the expression amount of tyrosinase is controlled by RBS of different strengths, thereby regulating the modification degree of DOPA, and finally obtaining high-yield mussel mucin with different DOPA modification degrees.

[0006] The first object of the present invention is to provide a method for synthesizing mussel mucin, wherein a gene sequence encoding an amino acid sequence as shown in any one of SEQ ID NO.2-6 is introduced into a plasmid to form a recombinant plasmid, the recombinant plasmid is introduced into a host bacterium to form a recombinant bacterium, the recombinant bacterium is inoculated into a fermentation medium for fermentation culture to obtain mussel mucin, and the amino acid sequence of the mussel mucin is shown in SEQ ID NO.12.

[0007] Furthermore, the amino acid sequence of SEQ ID NO.2 is obtained by deleting and mutating the amino acids at positions 2-3 of the N-terminus of the secondary structure of the mussel mucin shown in SEQ ID NO.1. The sequence of SEQ ID NO.2 is as follows:

[0008] MAVCIYFFLVGQIQAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0009] Furthermore, the amino acid sequence of SEQ ID NO.3 is obtained by deleting and mutating the amino acids at positions 2 to 12 of the N-terminus of the secondary structure of the mussel mucin shown in SEQ ID NO.1. The sequence of SEQ ID NO.3 is as follows:

[0010] MGQIQAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0011] Furthermore, the amino acid sequence of SEQ ID NO.4 is obtained by deleting and mutating the amino acids 2-14 at the N-terminus of the secondary structure of the mussel mucin shown in SEQ ID NO.1. The sequence of SEQ ID NO.4 is as follows:

[0012] MIQAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0013] Furthermore, the amino acid sequence of SEQ ID NO.5 is obtained by deleting and mutating the amino acids at positions 2 to 16 at the N-terminus of the secondary structure of the mussel mucin shown in SEQ ID NO.1. The sequence of SEQ ID NO.5 is as follows:

[0014] MAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0015] Furthermore, the amino acid sequence of SEQ ID NO.6 is obtained by deleting and mutating the amino acids 2-19 at the N-terminus of the secondary structure of the mussel mucin shown in SEQ ID NO.1. The sequence of SEQ ID NO.6 is as follows:

[0016] MIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0017] Further, the sequence of SEQ ID NO.1 is as follows:

[0018] MISAVCIYFFLVGQIQAGVYIPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0019] Further, the sequence of SEQ ID NO.12 is as follows:

[0020] IPFEKPGQCPVTRGITPCVCIPENSECRFDSNCPGAMKCCDFGCGCNKRCVPPVPSPLQCYYNGQYYPIGHFPVDGCNTCYCNDDGTVMCTLKACGYGYK

[0021] Furthermore, the recombinant plasmid also includes a gene sequence of tyrosinase, and the amino acid sequence of the tyrosinase is shown in SEQ ID NO.11.

[0022] Further, the sequence of SEQ ID NO.11 is as follows:

[0023] MAKYHRLNLQNPAAAPFLESYKKAITVMLQLPPSDARNWYRNAFIH

[0024] TLDCPHGNWWFVVWHRGYTGWFERTVRELSGDPNFAFPYWDWTALPQ

[0025] VPDSFFNGVLDPNNPAFIASYNEFYSQLSNPMSALWNSFSTAQLQQMRNR

[0026] GFQSVNDVWQAVRDSPMFFPRGRARTLTRQNPGFDATTRRAVSIGTIRNA

[0027] LAPTDFITFGSGKTANHSESATQGILESQPHNNVHNNIGGFMQDLLSPTDP

[0028] VFFAHHSNIDRLWDVWTRKQQRLGLPTLPTGANLPLWANEPFLFFIGPDG

[0029] KPVAKNKAGDYATIGDFDYNYEPGSGEAVIPAASRPGEMNNK

[0030] Furthermore, the expression of the tyrosinase is regulated by the RBS sequence shown in the gene sequence such as SEQ ID NO.7-10.

[0031] SEQ ID NO.7: AAGGGCCGAATT

[0032] SEQ ID NO.8: AAGGGGAGAGCC

[0033] SEQ ID NO.9: AAGGTGGTTCAT

[0034] SEQ ID NO.10:AAGAAGGGTTCATAG

[0035] Further, the plasmids include pUC18, pUC19, pGEM-T, pBR322, pET-28a, pET-21b, pBAD, pBBR1MCS-2, pBBR1MCS-5, pSC101, pMAL-c2 and pMAL-p2.

[0036] Furthermore, the host bacteria include Escherichia coli DH5α, Escherichia coli BL21 (DE3), Escherichia coli MG1655, Escherichia coli W3110, Escherichia coli TOP10, and Escherichia coli Nissle 1917.

[0037] Furthermore, the fermentation culture temperature is 20-45°C.

[0038] Furthermore, the pH of the fermentation culture is 4-7.

[0039] Furthermore, the fermentation medium includes a carbon source, a nitrogen source, an inorganic salt and metal ions.

[0040] Furthermore, the inoculation amount of the recombinant bacteria is 1%-5% by volume.

[0041] Beneficial effects of the present invention:

[0042] The present invention increases the yield of mussel mucin by performing deletion mutation on the N-terminal sequence of the secondary structure of mussel mucin of the amino acid sequence such as SEQ ID NO.1. The yield of mussel mucin in the prior art is about 2.57 g / L, while the maximum yield of the mussel mucin of the present invention is about 5 g / L, which is greatly improved compared with the yield of mussel mucin in the prior art. On this basis, mussel mucin is DOPA-modified by co-expressing tyrosinase, and the expression amount of tyrosinase is controlled by RBS of different strengths, thereby regulating the modification degree of DOPA, realizing the controllable modification of DOPA in mussel mucin while increasing the yield of mussel mucin, and finally obtaining high-yield mussel mucin with different DOPA modification degrees. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0044] Figure 1 It is the protein secondary structure of mussel mucin;

[0045] Figure 2is the effect of the N-terminal deletion of mussel mucin on its expression level, wherein pvfp1 represents a recombinant strain containing pET32a-pvfp; pvfp2 represents a recombinant strain containing pET32a-pvfp-del3; pvfp3 represents a recombinant strain containing pET32a-pvfp-del12; pvfp4 represents a recombinant strain containing pET32a-pvfp-del14; pvfp5 represents a recombinant strain containing pET32a-pvfp-del16; pvfp6 represents a recombinant strain containing pET32a-pvfp-del19;

[0046] Figure 3 Effects of different strengths of RBS regulating tyrosinase expression on the degree of DOPA modification of mussel mucin;

[0047] Figure 4 It is the mussel mucin yield of recombinant Escherichia coli fermented in a 5L fermenter. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0049] Bacterial strain: Escherichia coli Nissle 1917;

[0050] Plasmid: pET32a;

[0051] LB medium: yeast powder 5g / L, tryptone 10g / L, sodium chloride 10g / L;

[0052] TB medium: tryptone: 24 g / L, yeast extract: 24 g / L, glycerol: 4 mL / L, potassium phosphate (K2HPO4): 9.4 g / L, potassium dihydrogen phosphate (KH2PO4): 2.2 g / L;

[0053] Preparation of competent E. coli: First, pick a single colony from a fresh plate, inoculate it into 5 mL of LB medium, and culture it at 37°C overnight with shaking. The next day, take 1 mL of overnight cultured bacteria and add it to 100 mL of fresh LB medium, and culture it at 37°C with shaking until OD 600It is about 0.4-0.6. Transfer the culture solution to a centrifuge tube, centrifuge at 4°C and 4000rpm for 10 minutes, and discard the supernatant. Resuspend the bacteria with ice-cold 0.1M CaCl2 solution to a final volume of about 1 / 10 of the original culture volume, and then place the bacterial solution on ice for 30 minutes. Centrifuge again at 4°C and 4000rpm for 10 minutes, discard the supernatant, and resuspend the bacteria with ice-cold 0.1M CaCl2 solution to a final volume of about 1 / 100 of the original culture volume. Finally, divide the resuspended bacterial solution into sterile 1.5mL centrifuge tubes, about 100μL per tube, and quickly store them in a -80°C refrigerator for subsequent transformation experiments.

[0054] Purification of mussel foot protein: First, collect the bacteria and centrifuge at 10,000 rpm for 5 minutes. Take the bacterial precipitate and wash it with saline to remove impurities. Then, the washed cells are mechanically broken or ultrasonically treated to release the mucin. The broken material is added to an acidic solution to promote the dissolution of the mucin. Subsequently, the mussel mucin is separated and purified by affinity chromatography, and the purity is improved by combining precipitation, filtration and other techniques. The purified mucin solution needs to be concentrated and dried to obtain a powdered product.

[0055] Determination of mussel foot protein production: First, prepare standard protein solutions and sample solutions of different concentrations; then, add Coomassie Brilliant Blue G-250 dye to each sample and standard, mix well and incubate at room temperature for 5-15 minutes; then, use a spectrophotometer to measure the absorbance at 595nm; finally, calculate the protein content in the sample based on the standard curve.

[0056] Determination of DOPA modification degree: Preparation of standard samples: prepare a series of DOPA standard solutions with known concentrations. Measure the absorbance: use a UV-visible spectrometer to measure the absorbance of samples and standards at a wavelength of 280nm. Data analysis: draw a standard curve based on the absorbance of the standard and the known concentration. Calculate the DOPA content in the sample by comparing the absorbance of the sample.

[0057] Example 1: Construction of recombinant Escherichia coli producing mussel mucin

[0058] (1) Deletion optimization of the N-terminal sequence of mussel mucin

[0059] According to the genomic sequence of P. perna mussel mucin, the gene pvfp was synthesized by GENEWIZ (Suzhou), and the amino acid sequence of the secondary structure of mussel mucin pvfp is shown in SEQ ID NO.1 (GenBank: UNW37542.1). The N-terminal sequence in the secondary structure not only participates in the translation initiation process of the protein, but also affects its stability. In the translation initiation stage, the sequence near the ribosome binding site (RBS) and the start codon (AUG) in the N-terminal sequence determines the translation efficiency. Optimizing these sequences can significantly improve the expression level of the protein. Therefore, according to the secondary structure of mussel mucin (such as Figure 1 As shown), primers were designed to delete the N-terminal amino acid sequence of the secondary structure of mussel mucin. The synthetic pvfp gene sequence was used as a template, and primers pvfp-del3-F and pvfp-del3-R, pvfp-del12-F and pvfp-del12-R, pvfp-del14-F and pvfp-del14-R, pvfp-del16-F and pvfp-del16-R, pvfp-del19-F and pvfp-del19-R were designed for PCR amplification to obtain the pvfp-del3, pvfp-del12, pvfp-del14, pvfp-del16 and pvfp-del19 gene sequences, and then the pvfp-del3, pvfp-del12, pvfp-del14, pvfp-del16 and pvfp-del19 gene sequences were obtained by T5 exonuclease-dependent The pvfp, pvfp-del3, pvfp-del12, pvfp-del14, pvfp-del16 and pvfp-del19 gene sequences were connected to plasmid pET32a by the TEDA method to construct recombinant expression plasmids pET32a-pvfp, pET32a-pvfp-del3, pET32a-pvfp-del12, pET32a-pvfp-del14, pET32a-pvfp-del16 and pET32a-pvfp-del19.

[0060] (2) Construction of the mussel mucin synthesis pathway in Escherichia coli

[0061] The recombinant plasmids pET32a-pvfp, pET32a-pvfp-del3, pET32a-pvfp-del12, pET32a-pvfp-del14, pET32a-pvfp-del16 and pET32a-pvfp-del19 were transformed into Escherichia coli Nissle1917 by chemical transformation to obtain recombinant strains pvfp1-6 (where pvfp1 represents the recombinant plasmid containing pET32a-p vfp; pvfp2 represents the recombinant strain containing pET32a-pvfp-del3; pvfp3 represents the recombinant strain containing pET32a-pvfp-del12; pvfp4 represents the recombinant strain containing pET32a-pvfp-del14; pvfp5 represents the recombinant strain containing pET32a-pvfp-del16; pvfp6 represents the recombinant strain containing pET32a-pvfp-del19). Select a single colony, inoculate it into 5 mL LB medium, and culture it at 37°C overnight. Inoculate it into 25 mL TB medium with an inoculum of 1% and wait until it grows to OD 600 =1 was induced by adding 0.2 mM IPTG, and the cells were collected by centrifugation after 48 h of fermentation, and the mussel mucin was purified and the mussel mucin content was determined (e.g. Figure 2 shown).

[0062] Table 1 Primers used for N-terminal deletion of mussel mucin gene pvfp-6

[0063] Primer Name Sequence (5’-3’) Sequence ID pvfp-del3-F AGGAGATATACATATGGCAGTTTGTATATATTTCT SEQ ID NO.13 pvfp-del3-R ATATATACAAACTGCCATATGTATATCTCCT SEQ ID NO.14 pvfp-del12-F AGGAGATATACATATGGGCCAGATACAGGCTGG SEQ ID NO.15 pvfp-del12-R CCAGCCTGTATCTGGCCCATATGTATATCTCCT SEQ ID NO.16 pvfp-del14-F AGGAGATATACATATGCATACCATTTGAAAAACCCG SEQ ID NO.17 pvfp-del14-R CGGGTTTTTCAAATGGTATGCATATGTATATCTCCT SEQ ID NO.18 pvfp-del16-F AGGAGATATACATATGATTTGAAAAACCCGGACAGTG SEQ ID NO.19 pvfp-del16-R CACTGTCCGGGTTTTTCAAATCATATGTATATCTCCT SEQ ID NO.20 pvfp-del19-F AGGAGATATACATATGACCCGGACAGTGTCCAGTC SEQ ID NO.21 pvfp-del19-R GACTGGACACTGTCCGGGTCATATGTATATCTCCT SEQ ID NO.22

[0064] Example 2: Control of the degree of modification of mussel mucin DOPA

[0065] (1) Construction of a controllable DOPA-modified mussel system in Escherichia coli

[0066] According to the genome sequence of V. spinosum tyrosinase, the gene Vs-tyr was synthesized by GENEWIZ (Suzhou), and the amino acid sequence of Vs-tyr is shown in SEQ ID NO. 11. Using the synthesized Vs-tyr gene sequence as a template, primers Vs-tyr-RBS1-F, Vs-tyr-RBS2-F, Vs-tyr-RBS3-F, Vs-tyr-RBS4-F and Vs-tyr-RBS-R were designed for PCR amplification, and the plasmid pET32a-pvfp-del16 of the recombinant strain pvfp5 with the highest yield was selected, and then the gene Vs-tyr connected with RBS of different strengths was connected by the TEDA method. The plasmid pET32a-pvfp-del16 was connected to construct the recombinant expression plasmids pET32a-pvfp-del16-Vs-tyr-RBS1, pET32a-pvfp-del16-Vs-tyr-RBS2, pET32a-pvfp-del16-Vs-tyr-RBS3 and pET32a-pvfp-del16-Vs-tyr-RBS4 of RBS with different strengths. (The sequences of RBS1-4 are shown in SEQ ID NO.7-10. According to the prediction of the website RBS Calculator, the strength is enhanced by 2 times in sequence)

[0067] Table 2 Primers used for optimization of tyrosinase Vs-tyr RBS expression

[0068]

[0069]

[0070] (2) Detection of DOPA modification degree of mussel mucin produced by recombinant Escherichia coli

[0071] The constructed recombinant plasmids pET32a-pvfp-del16-Vs-tyr-RBS1, pET32a-pvfp-del16-Vs-tyr-RBS2, pET32a-pvfp-del16-Vs-tyr-RBS3 and pET32a-pvfp-del16-Vs-tyr-RBS4 were transformed into Escherichia coli Nissle1917 by chemical transformation to obtain recombinant strains pvfp7-10 (wherein, pvfp7 represents the recombinant strain containing pET32a-pvfp-del16-Vs-tyr-RBS1; pvfp8 represents the recombinant strain containing pET32a-pvfp-del16-Vs-tyr-RBS2; pvfp9 represents the recombinant strain containing pET32a-pvfp-del16-Vs-tyr-RBS3; pvfp10 represents the recombinant strain containing pET32a-pvfp-del16-Vs-tyr-RBS4). Select a single colony, inoculate it into 5 mL LB medium, and culture it at 37°C overnight. Inoculate it into 25 mL TB medium with an inoculum size of 1% and wait for it to grow to OD 600 = 1, 0.2 mM IPTG was added for induction, and the cells were collected by centrifugation after 48 h of fermentation. The mussel mucin was purified and the degree of DOPA modification of the mussel mucin was determined (e.g. Figure 3 shown).

[0072] Example 3: 5 L Fermenter Fed-Batch Fermentation of Recombinant E. coli Mussel Mucin

[0073] 5L fermentation tank fermentation production: The recombinant strain pvfp10 constructed in Example 2 was inoculated into 5mL LB medium and cultured overnight at 220rpm and 37°C. The seed solution was 600 =0.1 was transferred to a baffled Erlenmeyer flask with 25mL fermentation medium, and cultured at 220rpm and 30℃ for 10h, then inoculated into a 5L fermenter with 10% inoculum. The initial temperature was set at 37℃, the rotation speed was 3000r / min, and after 4h of fermentation, IPTG was added with a final concentration of 0.2mM to induce gene expression. During the fermentation process, 14% ammonia water was used to control the pH of the fermentation liquid at around 7, and glycerol was added to maintain the glycerol content in the fermenter at around 1%. Figure 4 It can be seen that the yield of mussel mucin obtained by fermentation of the recombinant strain pvfp10 in a 5L fermenter is 5g / L (e.g. Figure 4 shown).

[0074] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for synthesizing mussel mucin, characterized in that: The gene sequence encoding the amino acid sequence as shown in any one of SEQ ID NO.2-6 is introduced into a plasmid to form a recombinant plasmid, the recombinant plasmid is introduced into a host bacterium to form a recombinant bacterium, the recombinant bacterium is inoculated into a fermentation medium for fermentation culture to obtain mussel mucin, and the amino acid sequence of the mussel mucin is shown in SEQ ID NO.

12.

2. The method according to claim 1, characterized in that The recombinant plasmid also includes the gene sequence of tyrosinase.

3. The method according to claim 2, characterized in that The tyrosinase is expressed under the regulation of the RBS sequence shown in SEQ ID NO. 7-10.

4. The method according to claim 2, characterized in that: The amino acid sequence of the tyrosinase is shown in SEQ ID NO.

11.

5. The method according to claim 1, characterized in that The plasmids include pUC18, pUC19, pGEM-T, pBR322, pET-28a, pET-21b, pBAD, pBBR1MCS-2, pBBR1MCS-5, pSC101, pMAL-c2, and pMAL-p2.

6. The method according to claim 1, characterized in that The host bacteria include Escherichia coli DH5α, Escherichia coli BL21 (DE3), Escherichia coli MG1655, Escherichia coli W3110, Escherichia coli TOP10, and Escherichia coli Nissle 1917.

7. The method according to claim 1, characterized in that The fermentation temperature is 20-45°C.

8. The method according to claim 1, characterized in that The pH of the fermentation culture is 4-7.

9. The method according to claim 1, characterized in that: The fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt and metal ions.

10. The method according to claim 1, characterized in that The inoculation amount of the recombinant bacteria is 1%-5% by volume.

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