Construction method and fermentation preparation method of hybrid mussel protein

Abstract

The invention belongs to the field of genetic engineering and protein materials, and particularly relates to a construction method and a fermentation preparation method of heterozygous mussel protein. The 3-type and 5-type mussel proteins are successfully expressed in escherichia coli, a series of double-fragment and three-fragment hybrid mussel proteins are designed and constructed based on the 3-type and 5-type mussel proteins, and finally escherichia coli genetically engineered bacteria for preparing high-viscosity mussel proteins are screened out. The genetically engineered bacterium is subjected to fermentation process optimization, the OD600 reaches 55-65 after the genetically engineered bacterium is fermented for 12-14 hours in a 5L fermentation tank, and the yield can reach 1.5-2.0 g / L, which is 18-25 times that of shake flask fermentation. The method provided by the invention has the advantages of strong universality, short time, low cost, high expression efficiency and the like, and lays a foundation for industrial production of mussel protein.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and protein materials, and in particular to a construction method and a fermentation preparation method of a hybrid mussel protein. Background Art

[0002] Mussel foot proteins (mfps), also known as mussel adhesive proteins, are adhesives secreted by the byssus of marine mussels such as the Mediterranean mussel (Mytilus galloprovincalis), the thick-shelled mussel (Mytilus coruscus), and the green mussel (Perna viridis). Currently, six types of mfps (mfp-1 to mfp-6) have been discovered. Mussel foot proteins have the characteristics of a wide adhesion range, high strength, and good bioaffinity. They are important biomimetic objects and sources of innovative strategies for studying biological wet adhesion.

[0003] The extraction of natural mussel protein is very difficult. Genetic engineering provides a new method for the production of mussel protein. Mfps have achieved heterologous expression in both eukaryotic and prokaryotic hosts. However, there are still some problems with recombinant Mfps, such as low yield caused by toxic soluble expression, low purification efficiency, poor solubility, etc. In order to overcome these defects, researchers have constructed a variety of new hybrid mussel proteins composed of fp-1, fp-3 and fp-5. The protein is mainly expressed as inclusion bodies, and the longer coding sequence occupies more translation resources, resulting in higher yields of the three-fragment hybrid proteins fp-151, fp-131 and fp-353 than single proteins. fp-151 was cultured in a 200L fed-batch bioreactor and obtained a recombinant mussel protein yield of about 1g / L. Some researchers have also optimized the purification method to obtain a pure product rate of 0.7g / L fp-151, which is the highest expression and purification yield known so far. In addition, among the different two-fragment fusion proteins fp-33, fp-35, and fp-31, the fusion protein composed of fp-3 and fp-5 has the best adhesion performance. This may be because the type 3 and type 5 proteins themselves have excellent adhesion and therefore show the strongest adhesion after combination. However, for the type 3 and type 5 proteins with the best adhesion, there are no reports on the series design and mass production of fusion proteins.

[0004] Therefore, it is of great significance to develop a new high-viscosity and high-yield mussel protein production process based on multi-fragment hybridization of mussel protein. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a hybrid mussel protein and its construction method and fermentation preparation method in view of the deficiencies of the prior art. The present invention adopts genetic engineering technology to construct a plasmid of tandem multi-copy mussel protein, uses Escherichia coli as a host, expresses and obtains different series of hybrid mussel proteins, provides a method for comparing the adhesion properties of the above hybrid mussel proteins, provides the optimization of the fermentation process of the above hybrid mussel proteins, and realizes the high-level production of highly viscous mussel proteins.

[0006] In order to solve the above technical problems, the present invention discloses a hybrid mussel protein and a construction method and a fermentation preparation method thereof. The specific technical scheme is as follows:

[0007] A hybrid mussel protein comprising one type 3 mussel protein and one or two type 5 mussel proteins. Preferably, the hybrid mussel protein is any one of the following a1 to a4:

[0008] a1, containing type 5 mussel protein, type 3 mussel protein, and type 5 mussel protein (fp-535) from N-terminus to C-terminus;

[0009] a2, containing mussel protein type 5, mussel protein type 5, and mussel protein type 3 (fp-553) from N-terminus to C-terminus;

[0010] a3, containing type 3 mussel protein and type 5 mussel protein (fp-35) from N-terminus to C-terminus;

[0011] a4, contains mussel protein type 5 and mussel protein type 3 (fp-53) from N-terminus to C-terminus.

[0012] Wherein, the type 3 mussel protein is the type 3B protein Mgfp-3B or the mutant 3B of Mgfp-3B from the Mediterranean mussel Mytilus galloprovincalis m ; The Mediterranean mussel is a wild type Mediterranean mussel. Preferably, the 3B m The invention comprises single mutants and combined mutants of Mgfp-3B, wherein the single mutant is any one of G33Y, N43Y or N76Y, and the combined mutant is any one of G33Y / N43Y, N43Y / N76Y, G33Y / N76Y or G33Y / N43Y / N76Y obtained by two-two mutation and three-three mutation. m The specific amino acid sequence and nucleotide sequence of are detailed in the Chinese patent with patent number CN115181169A. More preferably, the type 3 mussel protein is 3B m The combined mutant G33Y / N43Y / N76Y showed the best adhesion.

[0013] The type 5 mussel protein is derived from wild-type Mediterranean mussel Mytilus galloprovincalis and is named Mgfp-5. Its amino acid sequence is shown in SEQ ID NO.1.

[0014] Among them, the amino acid sequence of the hybrid mussel protein shown by a1 to a4 is shown in SEQ ID NOs. 2 to 5, and the coding gene sequence is shown in SEQ ID NOs. 6 to 9.

[0015] In a second aspect, the present invention provides an expression cassette or a recombinant expression vector, wherein the expression cassette or the expression vector contains the coding gene sequence of the hybrid mussel protein described in the first aspect. Preferably, the recombinant expression vector is pet28a-Mgfp-3B, pet28a-Mgfp-3B m (fp-3), pet28a-Mgfp-5(fp-5), pet28a-Mgfp-35(fp-35), pet28a-Mgfp-53(fp-53 ), pet28a-Mgfp-535(fp-535), pet28a-Mgfp-553(fp-553) or pACYCDuet-Mgfp-535.

[0016] In a third aspect, the present invention provides a recombinant Escherichia coli containing the expression cassette or recombinant expression vector described in the second aspect. Preferably, the starting bacteria of the recombinant bacteria are Escherichia coli BL21 (DE3).

[0017] In a fourth aspect, the present invention provides a method for preparing the hybrid mussel protein according to the first aspect, comprising the following steps:

[0018] (1) amplifying the coding gene sequence of the hybrid mussel protein and introducing it into an expression vector to construct a recombinant expression vector; and introducing the recombinant expression vector into Escherichia coli to obtain a recombinant bacterium;

[0019] (2) inoculating the seed liquid of the recombinant bacteria into a fermentation medium for fermentation to obtain a fermentation liquid, and extracting and purifying the fermentation liquid to obtain a hybrid mussel protein.

[0020] Wherein, in step (1), the expression vector is any one of pet28a, pACYCDuet-1, pETDuet-1, and pRSFDuet-1; one or two copies of the gene encoding the hybrid mussel protein are introduced into the recombinant expression vector. Preferably, the specific method of step (1) is as follows: the fp-5 protein gene is synthesized by General Biosystems (Anhui) Co., Ltd. and connected to the pet28a(+) vector to obtain the pet28a-fp-5 plasmid, and then,

[0021] (1) The fp-5 gene was isolated from pet28a-fp-5 by restriction endonucleases Sal I and Xho I, and ligated into pet28a-fp-3 digested with Xho I to obtain plasmid pet28a-fp-35;

[0022] (2) The fp-3 gene was isolated from pet28a-fp-3 by restriction endonucleases Sal I and Xho I, and ligated into pet28a-fp-5 digested with Xho I to obtain plasmid pet28a-fp-53;

[0023] (3) The fp-35 gene was isolated from pet28a-fp-35 by restriction endonucleases Sal I and Xho I, and ligated into pet28a-fp-5 digested with Xho I to obtain plasmid pet28a-fp-535;

[0024] (4) The fp-53 gene was isolated from pet28a-fp-53 by restriction endonucleases Sal I and Xho I, and ligated into pet28a-fp-5 digested with Xho I to obtain plasmid pet28a-fp-553;

[0025] (5) Using pET28a-fp-535 as a template, the fp-535 fragment was amplified and ligated into the pACYCDuet-1 vector digested with Nco I and Eco RI to obtain the first plasmid, which was then digested with Nde I and Kpn I to obtain the first linear vector. Using pET28a-fp-535 as a template, the fp-535 fragment was amplified and ligated into the first linear vector to obtain the plasmid pACYCDuet-2×fp535 containing two fp-535 fragments.

[0026] The obtained plasmids were transformed into the expression host Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0027] Wherein, in step (2), the inoculation volume ratio is 10%-20%; the fermentation culture, the fermentation temperature is 32-37°C, the fermentation pH is 6.5-7.5, the total fermentation time is 12-16h, the dissolved oxygen is 20-40%, the ventilation volume is 2-5L / min, and the fermentation is carried out until the OD of the bacterial liquid reaches 600 When the pH is 30 to 50, 0.1 to 1.2 mM isopropyl-β-D-thiogalactoside IPTG is added to induce fermentation for 4 to 8 hours; when the carbon source in the initial fermentation medium is exhausted, feed medium is added at a flow rate of 30 to 60 mL / h. Preferably, the seed solution of the recombinant bacteria is obtained by inoculating the recombinant bacteria into the seed medium and amplifying the culture step by step.

[0028] Wherein, the fermentation medium comprises a carbon source, a nitrogen source and an inorganic salt; preferably, the carbon source is any one or more of glucose, glycerol, sucrose, lactose, galactose, fructose, xylose and water-soluble starch; the nitrogen source is any one or more of peptone, yeast powder, beef extract and corn steep liquor; the inorganic salt is any one or more of magnesium sulfate and potassium dihydrogen phosphate;

[0029] Preferably, the formula of the fermentation medium is: 10-30 g / L glycerol, 20-40 g / L yeast powder, 10-30 g / L peptone, 0.5-2 g / L magnesium sulfate, 2-8 g / L potassium dihydrogen phosphate;

[0030] The feed culture medium is a 50-500 g / L glucose aqueous solution.

[0031] Preferably, in step (2), the extraction is column chromatography or acid extraction, preferably column chromatography.

[0032] Among them, the specific method of column chromatography is to resuspend the fermented bacteria with a lysis buffer (0.1mol / L NaH2PO4, 0.01mol / L Tris-Cl, 8mol / L urea, pH=8.0), collect the supernatant solution after ultrasonic disruption, combine the filtrate with a HisTrap HP chromatography column after passing through a membrane, wash impurities with a washing buffer (0.1mol / L NaH2PO4, 0.01mol / L Tris-Cl, 8mol / L urea, pH=5.9), then collect the target protein with an elution buffer (0.5mol / L HCl), and finally dialyze with 5% (v / v) acetic acid and freeze-dry.

[0033] The specific method of acid extraction is to dissolve the fermented bacteria in PBS buffer, collect the precipitate after ultrasonic disruption, first extract the mussel protein with 25% (v / v) acetic acid overnight, and finally dialysis and purification with 5% (v / v) acetic acid and freeze-dry.

[0034] Furthermore, the present invention also provides a method for comparing the adhesion performance of mussel proteins, which specifically includes a glass lap experiment and AFM imaging.

[0035] The glass lap test steps are as follows: 5 mg of purified mussel protein is dissolved in 10 μL of 5% (v / v) acetic acid, and the mixture is evenly applied to the top of the glass substrate and bonded overnight at 37° C. The lap shear test is performed using a universal testing machine.

[0036] Among them, the AFM imaging experimental steps are: dissolve the purified mussel protein in 5% (v / v) acetic acid at a final concentration of 1 mg / mL, take 10 μL and drop it on the mica surface, and after drying at room temperature, use AFM to scan the height map and phase map of the protein-coated mica sheet. Then rinse the mica sheet in pure water for 4 hours, and observe the protein remaining on the mica surface in the same way. Use NanoScope software to calculate the particle coverage and roughness of the mica surface before and after rinsing. Preferably, the hybrid mussel protein with the strongest adhesion performance and coating force is fp-535, and the adhesion strength reaches 1.43MPa.

[0037] In a fifth aspect, the present invention provides the use of the hybrid mussel protein described in the first aspect in daily skin care, biological adhesion and waterproof coating.

[0038] Beneficial effects:

[0039] The present invention provides a hybrid mussel protein and its construction and fermentation preparation method. The expression amount and viscosity of mussel protein are enhanced by connecting multiple protein fragments, and the fermentation process is optimized in shake flasks and fermentation tanks, so as to realize the high-density fermentation process of Escherichia coli to efficiently synthesize high-viscosity mussel protein. The hybrid protein fp-535 has excellent coating power, and the adhesion strength reaches 1.43MPa. The OD of 1.044g / L is 0.044g / L in a 5L fermentation tank for 12 to 14 hours. 600 When the concentration reaches 55-65, the yield can reach 1.5-2.0 g / L. The fermentation process and purification process established by the invention lay a foundation for the industrial production of mussel protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0041] Figure 1 This is the SDS-PAGE analysis of the expression of each mussel protein in Example 1 of the present invention. Lane M is the protein marker, lane S is the soluble part of the protein, and lane IS is the insoluble part of the protein.

[0042] Figure 2 The figure is a comparison of the expression of various mussel proteins and cell growth in Example 1 of the present invention.

[0043] Figure 3 The figure is a comparison of the bonding strength of each mussel protein in Example 2 of the present invention.

[0044] Figure 4 The AFM height map and phase diagram changes of mussel protein adsorption in Example 2 of the present invention.

[0045] Figure 5The roughness change of mussel protein adsorbed on the mica surface in Example 2 of the present invention.

[0046] Figure 6 The coverage of mussel protein particles adsorbed on the mica surface in Example 2 of the present invention is changed.

[0047] Figure 7 This is the SDS-PAGE analysis of fp-535 expression in Example 3 of the present invention. Lane M is the protein marker, lane S is the soluble part of the protein, and lane IS is the insoluble part of the protein.

[0048] Figure 8 This is the SDS-PAGE analysis of fp-535 expression under different fermentation conditions in Example 4 of the present invention.

[0049] Fig. 9 This is the SDS-PAGE analysis of fp-535 expression under different culture medium components in Example 4 of the present invention.

[0050] Fig.10 This is the SDS-PAGE analysis of fp-535 expression at different induction times in Example 5 of the present invention. 4, 5, 6, 7, and 8 hours are the culture times after induction.

[0051] Fig.11 The figure is a comparison of parameters during the fermentation process at different induction times in Example 5 of the present invention.

[0052] Fig.12 This is the process curve of the hybrid protein fp-535 in the 5L fermenter in Example 5 of the present invention. DETAILED DESCRIPTION

[0053] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0054] Example 1 Construction and expression of fusion mussel protein

[0055] In the early stage of the laboratory, single mutations were performed on the wild-type Mediterranean mussel 3B protein, and single mutants G33Y, N43Y and N76Y were obtained in sequence. The specific method refers to the method for constructing the Mgfp-3B mutant described in Example 3 of the Chinese patent with patent number CN115181169A.

[0056] In this example, the mutation sites of the wild-type Mediterranean mussel 3B protein obtained above were further combined in pairs and three-by-three mutations, and G33Y / N43Y, N43Y / N76Y, G33Y / N76Y, and G33Y / N43Y / N76Y combination mutants were obtained in sequence. The specific construction process of the combination mutants can refer to the construction process of the single mutants in patent CN115181169A.

[0057] The single mutants and combined mutants of wild-type Mediterranean mussel 3B protein are collectively referred to as mutant 3B m .

[0058] The adhesion of the mussel protein obtained after the expression, fermentation and purification of the above-mentioned combined mutants was measured by glass lap experiment, and the results are shown in Table 1. Among them, the glass lap experiment, the expression and fermentation and purification of the combined mutants can refer to the experimental process in patent CN115181169A.

[0059] Table 1 Adhesion of combined mutants of wild-type Mediterranean mussel 3B protein

[0060]

[0061] The results showed that the wild-type Mediterranean mussel 3B protein with triple mutation had the best adhesion. Subsequent experiments were carried out with the mutant G33Y / N43Y / N76Y of the wild-type Mediterranean mussel 3B protein, and the expression vector was pet28a-fp-3.

[0062] The wild-type Mediterranean mussel protein fp-5 protein gene (amino acid sequence as shown in SEQ ID NO.1) was synthesized by General Biosystems (Anhui) Co., Ltd. and ligated to the pet28a(+) vector to obtain the pet28a-fp-5 plasmid.

[0063] Based on plasmids pET28a-FP-3 and pET28a-FP-5, the following four plasmids containing hybrid mussel proteins were constructed:

[0064] (1) The fp-5 gene was isolated from pet28a-fp-5 by restriction endonucleases Sal I and Xho I, and ligated into pet28a-fp-3 digested with Xho I to obtain plasmid pet28a-fp-35, wherein the plasmid pet28a-fp-35 contained a hybrid mussel protein fp-35, the amino acid sequence of which was shown in SEQ ID NO.4, and the nucleotide sequence of which was shown in SEQ ID NO.8.

[0065] (2) Similarly, the fp-3 gene was isolated from pet28a-fp-3 using restriction endonucleases Sal I and Xho I, and ligated into pet28a-fp-5 digested with Xho I to obtain plasmid pet28a-fp-53, wherein the plasmid pet28a-fp-53 contains the hybrid mussel protein fp-53, whose amino acid sequence is shown in SEQ ID NO.5 and whose nucleotide sequence is shown in SEQ ID NO.9.

[0066] (3) Next, the fp-35 gene was isolated from pet28a-fp-35 using restriction endonucleases Sal I and Xho I, and ligated into pet28a-fp-5 digested with Xho I to obtain plasmid pet28a-fp-535, wherein the plasmid pet28a-fp-535 contains the hybrid mussel protein fp-535, whose amino acid sequence is shown in SEQ ID NO.2 and whose nucleotide sequence is shown in SEQ ID NO.6.

[0067] (4) The fp-53 gene was isolated from pet28a-fp-53 by restriction endonucleases Sal I and Xho I, and ligated into pet28a-fp-5 digested with Xho I to obtain plasmid pet28a-fp-553, wherein the plasmid pet28a-fp-553 contained the hybrid mussel protein fp-553, whose amino acid sequence was shown in SEQ ID NO.3 and whose nucleotide sequence was shown in SEQ ID NO.7.

[0068] The plasmids constructed above were transformed into the expression host E. coli BL21 (DE3). Single clones were picked and cultured in LB medium at 37°C overnight. The bacterial solution was expanded at a ratio of 1:1000. 600 When the pH value was 0.6 to 0.8, IPTG was added at a final concentration of 1 mM for induction. After induction at 37°C and 200 rpm for 6 hours, 1.0 mL of bacterial solution was collected, the precipitate was resuspended in PBS (pH 7.4), ultrasonically broken on ice until the solution was clear, centrifuged at 12000 r / min for 20 minutes, the supernatant was taken, the precipitate was resuspended in the same amount of PBS, and the expression form of the protein was analyzed by SDS-PAGE. The grayscale scanning calculation was performed by Gel-Pro Analyzer (Media Cybernatics, version 4.0.0.4) software, and the total protein content in the supernatant or precipitate was detected by Bradford method. The calculation method of mussel protein yield was the grayscale proportion of the target protein multiplied by the total protein concentration.

[0069] SDS-PAGE picture Figure 1As shown, fp-35 and fp-53 showed obvious bands at around 17 kDa, and fp-553 and fp-535 showed obvious bands at around 28 kDa, indicating the expression of the proteins. Figure 2 The results showed that the total production of hybrid mussel protein was higher than the expression level of fp-3 or fp-5, the expression levels of fp-35 and fp-53 were about 45 mg / L, and the expression levels of fp-553 and fp-535 were about 55 mg / L. In addition, the insoluble inclusion bodies formed by the fusion protein were non-toxic to cells and reduced the impact on cell growth. All these indicate that the increase of fusion fragments will promote the expression of mussel protein and the growth of host cells.

[0070] Example 2 Comparison of adhesion properties of mussel protein

[0071] The adhesion of mussel protein was analyzed by glass lap test: 5 mg of mussel protein purified by column chromatography was dissolved in 10 μL of 5% (v / v) acetic acid, evenly applied to the top of the glass substrate (the contact surface was about 10 mm wide × 10 mm long) and bonded at 37°C overnight. The lap shear test was performed using a universal testing machine, with a 100N load cell for force measurement and a speed of 10 mm / min in the shear direction. The shear strength was determined by dividing the maximum force by the bonding area.

[0072] AFM imaging was used to analyze the coating ability of mussel protein: the purified mussel protein was dissolved in 5% (v / v) acetic acid at a final concentration of 1 mg / mL, 10 μL was dropped on the mica surface, and after drying at room temperature, the height map and phase map of the protein-coated mica sheet were scanned by AFM. Then the mica sheet was rinsed in pure water for 4 hours, and the protein remaining on the mica surface was observed in the same way. The particle coverage and roughness of the mica surface before and after rinsing were calculated using NanoScope software.

[0073] Figure 3 The results showed that the adhesion of fp-3 and fp-5 was 0.376 and 0.351 MPa, and the adhesion of fp-35, fp-53, fp-553 and fp-535 was 0.87, 0.901, 1.417 and 1.432 MPa respectively. The adhesion of two-fragment fusion protein and three-fragment fusion protein was 2.4 times and 3.9 times that of single-fragment fusion protein respectively. Since fp-53 and fp-535 had better performance, they were selected as the two-fragment and three-fragment fusion proteins in subsequent studies. Figure 4 The results showed that mussel proteins can be adsorbed on the mica surface with good coating force, and as the fusion fragments increase, more proteins are adsorbed on the mica surface. After washing with water, fp-3 and fp-5 slightly adhere to the mica surface, while fp-53 and fp-535 have more residues on the mica surface. Figure 5 and Figure 6The particle coverage and roughness of the mica surface before and after washing were analyzed, which was consistent with the observed results. Macroscopic and microscopic adhesion analysis showed that the number of mussel protein tandem modules was positively correlated with adhesion performance, and fp-535 had the best adhesion and coating force. Therefore, fp-535 was selected in subsequent experiments to optimize the expression of mussel protein.

[0074] Example 3 Construction of BL21 / pACYCDuet-fp-2×535

[0075] Using pET28a-fp-535 as a template, the fp-535 fragment was amplified with the primer pair 1-F / 1-R in Table 2, and ligated into the pACYCDuet-1 vector digested with Nco I and Eco RI to obtain the first plasmid, and then the first plasmid was digested with Nde I and Kpn I to obtain the first linear vector. Using pET28a-fp-535 as a template, the fp-535 fragment was amplified with the primer pair 2-F / 2-R in Table 2, and ligated into the above-mentioned first linear vector to obtain the plasmid pACYCDuet-2×535 containing two fp-535 fragments. Finally, the plasmid was transformed into Escherichia coli BL21 (DE3), and the heterologous expression of the hybrid mussel protein was analyzed according to the culture method described in Example 1.

[0076] Table 2 Primers for plasmid construction

[0077]

[0078] Figure 7 It shows that fp-535 is successfully expressed in the pACYCDuet vector, which is divided into a soluble part and an insoluble part. Compared with the pet28a vector, the fp-535 protein band is more obvious in pACYCDuet. According to the protein content detection method described in Example 1, the mussel protein content of BL21 / pACYCDuet-fp-2×535 was 67.7 mg / L, and the protein content of BL21 / pet28a-fp-535 was 58.07 mg / L. BL21 / pACYCDuet-fp-2×535 was selected for subsequent experiments.

[0079] Example 4 Construction of shake flask fermentation of mussel protein

[0080] The strain BL21 / pACYCDuet-fp-2×535 was inoculated into 250 mL shake flask fermentation medium with a volume of 50 mL and cultured at 37°C until OD 600 When the p-value was 0.6-0.8, IPTG with a final concentration of 1 mM was added to induce expression for 6 h, and the expression level of the hybrid protein was studied by SDS-PAGE.

[0081] The formula of the shake flask fermentation medium is: 15g / L glucose, 20g / L yeast powder, and 10g / L peptone.

[0082] Under the above initial culture conditions, an induced temperature gradient was first set, with 16°C, 20°C, 24°C, 28°C, 32°C, 37°C, and 40°C as variables, to study the effects of different induction temperatures on the expression of recombinant proteins.

[0083] Under the condition of optimal induction temperature, in a 250mL shake flask, the liquid volume was set as 25mL, 30mL, 35mL, 40mL, 45mL, and 50mL to study the effect of different liquid volumes on the expression of recombinant protein.

[0084] Under the conditions of optimal induction temperature and liquid volume, a concentration gradient of inducer IPTG was set, with 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1mM, and 1.2mM as variables, to study the effects of different inducer concentrations on the expression of recombinant protein.

[0085] The carbon source, nitrogen source and inorganic salt were optimized while other components of the shake flask fermentation medium remained unchanged. Eight carbon sources (glucose, glycerol, sucrose, lactose, galactose, fructose, xylose and water-soluble starch), different carbon source concentrations (0, 5, 10, 15, 20, 25 and 30 g / L), four nitrogen sources (peptone, yeast powder, beef extract and corn steep liquor), two ions (Mg and Mg) were added to the fermentation medium. 2+ PO4 3- ) to explore the effects of different concentrations and ratios on cell growth and fp-535 production. 2+ PO4 3- Derived from MgSO4 and KH2PO4 respectively.

[0086] Figure 8 The results showed that fp-535 had more obvious target bands at induction temperatures of 24, 32, and 37°C; the highest protein yield was observed in the baffled bottle with the smallest liquid volume, indicating that reducing the liquid volume and increasing oxygen would enhance the expression of fp-535; the inducer concentration of 0.2 to 1.2 mM all prompted fp-535 to present obvious expression bands.

[0087] Fig. 9The results showed that fp-535 had a high expression level in carbon sources other than xylose. Glycerol was selected as a carbon source due to its cost advantage and few metabolic byproducts. The initial glycerol concentration of 10-30 g / L was conducive to protein expression. When a single nitrogen source was added during the fermentation process, the accumulation of protein in peptone reached the maximum, while the bacterial biomass was the largest in yeast powder (about 0.93 g / L), but the protein yield was low. Therefore, the two substances were compounded as nitrogen sources. The results showed that when the ratio of yeast powder and peptone was 2:1 and the total concentration of the two was 40 g / L, it was most conducive to the fermentation of mussel protein. Finally, the effects of different ions on fermentation were compared. When 2 g / LMgSO4 and 4 g / L KH2PO4 were added, the protein yield at the shake flask level was the highest, and the highest yield and bacterial biomass reached 91.13 mg / L and 0.91 g / L. The optimized culture medium formula will be used for subsequent high-density fermentation. The optimized culture medium formula is: 20g / L glycerol, 26.7g / L yeast powder, 13.3g / L peptone, 2g / L MgSO4, and 4g / L KH2PO4.

[0088] Example 5 High-density fermentation of mussel protein

[0089] The mussel protein production strain was inoculated and streaked on LB solid medium plates to obtain activated engineering strains. A single colony of the activated strain was picked and inoculated into a 50 mL shake flask containing 10 mL LB liquid medium for primary seed liquid culture, and then the primary seed liquid was inoculated into a 500 mL shake flask containing 100 mL LB liquid medium for secondary inoculation to obtain OD 600 The secondary seed solution was about 5, and then the secondary seed solution was inoculated into a 5L fermenter with 2.5L at a 10% inoculation rate, with an initial ventilation of 2L / min, initial stirring of 200rpm, and a culture temperature of 37°C. The pH was controlled at about 7 with 25% (v / v) ammonia water to start the culture, and the speed and air flow were adjusted to keep the dissolved oxygen at 20% to 40%. The induction time during the fermentation process was optimized. Specifically, when the OD 600 When the pH value reached 30, 40 or 50, 0.2 mM IPTG was added for induction culture at 37°C. When the initial carbon source in the culture medium was exhausted, 300 g / L glucose aqueous solution was added at a rate of 30-60 mL / h to supplement the carbon source. Samples were taken at regular intervals to examine the fermentation level of mussel protein.

[0090] Fig.10 It was shown that under different induction times, mussel protein showed obvious bands at 4 h of induction, indicating the continuous expression of hybrid mussel protein.

[0091] Fig.11 It shows that in OD 600When the induction started at 30, the fermentation period was the shortest (12 h), the feed medium consumption was the least (79.8 g glucose), and the yield of hybrid mussel protein was the highest (2 g / L), which had obvious advantages.

[0092] Fig.12 Shows OD 600 When the pH reaches 30, the fermentation process begins to be induced. The bacteria grow slowly in the initial stage, and the biomass increases rapidly in the exponential stage. As the initial carbon source is exhausted, the bacteria begin to utilize the supplemented glucose. After induction, the growth rate of the bacteria slows down, and the regulation of pH and dissolved oxygen reaches a stable state.

[0093] The present invention provides a method for constructing a hybrid mussel protein and a fermentation preparation method. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A hybrid mussel protein, characterized in that It contains one type 3 mussel protein and one or two type 5 mussel proteins.

2. The hybrid mussel protein according to claim 1, characterized in that The hybrid mussel protein is any one of the following a1 to a4: a1, containing type 5 mussel protein, type 3 mussel protein, and type 5 mussel protein from N-terminus to C-terminus; a2, containing type 5 mussel protein, type 5 mussel protein, and type 3 mussel protein from N-terminus to C-terminus; a3, containing type 3 mussel protein and type 5 mussel protein from N-terminus to C-terminus; a4, contains type 5 mussel protein and type 3 mussel protein from N-terminus to C-terminus.

3. The hybrid mussel protein according to claim 1, characterized in that The type 3 mussel protein is derived from the type 3B protein Mgfp-3B or a mutant 3B of Mgfp-3B of the Mediterranean mussel Mytilus galloprovincalis. m ; The type 5 mussel protein is derived from the Mediterranean mussel Mytilus galloprovincalis, and its amino acid sequence is shown in SEQ ID NO.

1.

4. The hybrid mussel protein according to claim 2, characterized in that The amino acid sequence of the hybrid mussel protein described in a1 to a4 is shown in SEQ ID NOs. 2 to 5.

5. The hybrid mussel protein according to claim 4, characterized in that The coding gene sequence of the hybrid mussel protein described in a1 to a4 is shown in SEQ ID NOs. 6 to 9.

6. An expression cassette or a recombinant expression vector, characterized in that: It contains the coding gene sequence of any one or several combinations of hybrid mussel proteins as described in claim 5.

7. A recombinant Escherichia coli containing the expression cassette or recombinant expression vector according to claim 6.

8. The method for preparing the hybrid mussel protein according to any one of claims 1 to 5, characterized in that: The steps include: (1) amplifying the coding gene sequence of the hybrid mussel protein and introducing it into an expression vector to construct a recombinant expression vector; introducing the recombinant expression vector into Escherichia coli to obtain a recombinant bacterium; preferably, the expression vector is any one of pet28a, pACYCDuet-1, pETDuet-1 or pRSFDuet-1, and one or two copies of the coding gene of the hybrid mussel protein are introduced into the recombinant expression vector; (2) inoculating the seed liquid of the recombinant bacteria into a fermentation medium for fermentation and culturing, and purifying the fermentation liquid after the fermentation to obtain the hybrid mussel protein; preferably, the inoculation volume ratio is 10%-20%, the fermentation temperature is 32-37°C, the fermentation pH is 6.5-7.5, the total fermentation time is 12-16h, the dissolved oxygen is 20-40%, and the OD of the bacterial liquid is 0.1%. 600 When the carbon source in the initial fermentation medium is exhausted, feed medium is added at a flow rate of 30 to 60 mL / h.

9. The preparation method according to claim 8, characterized in that: In step (2), the fermentation medium comprises a carbon source, a nitrogen source and an inorganic salt; Wherein, the carbon source is any one or more combinations of glucose, glycerol, sucrose, lactose, galactose, fructose, xylose and water-soluble starch; The nitrogen source is any one or more of peptone, yeast powder, beef extract and corn steep liquor powder; The inorganic salt is any one or more combinations of magnesium sulfate and potassium dihydrogen phosphate; Preferably, the formula of the fermentation medium is: 10-30 g / L glycerol, 20-40 g / L yeast powder, 10-30 g / L peptone, 0.5-2 g / L magnesium sulfate, 2-8 g / L potassium dihydrogen phosphate; The feed culture medium is a 50-500 g / L glucose aqueous solution.

10. Use of the hybrid mussel protein according to any one of claims 1 to 5 in the preparation of daily skin care products, bioadhesives and waterproof coatings.

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