A bicistronic translation-coupled expression vector and its application

Abstract

This invention discloses a bicistronic translation-coupled expression vector. The expression vector is constructed by inserting a molecular chaperone, a novel ribosome binding site, and the target protein gene into the pET-28a(+) plasmid. Furthermore, this invention constructs a genetically engineered bacterium using *E. coli* BL21(DE3) as the expression host. Utilizing the bicistronic translation-coupled expression vector, the co-expression of mussel protein Mgfp-3B with the molecular chaperones SUMO and / or TrxA is achieved, promoting the correct folding and translation of Mgfp-3B for expression in a soluble form. This invention enables a simple and rapid production of bioactive soluble mussel protein in *E. coli*, and the bicistronic expression vector avoids the enzymatic chaperone manipulation required for fusion expression. In addition, this invention, through optimized fermentation medium and fermentation process, achieves a soluble mussel protein yield of 200-300 mg / L, simplifying downstream purification processes.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering and biotechnology, specifically to a method for constructing a bicistronic translation-coupled expression vector using a molecular chaperone and mussel protein, and for synergistic expression to improve soluble expression levels. Background Technology

[0002] Marine mussels are distributed in various coastal countries. Their foot secretes byssal threads, allowing them to firmly adhere to surfaces even under high-intensity impacts and in moist water. Mussel foot proteins (Mfps) are considered to play a major adhesive role in these byssal threads. The strong adhesive force of Mfps gives mussels durable adhesion strength in water and allows them to maintain flexibility and elasticity on almost all types of organic and inorganic materials. The excellent properties of Mfps have attracted much attention in terms of protein composition, the hypothesis of adhesion mechanisms, and the development of Mfps-inspired adhesive materials.

[0003] To further study mussel proteins, a certain amount of protein must be obtained. Using direct extraction methods, extracting 1g of mussel foot protein (a mixture of Mfp-1 and Mfp-2) requires tens of thousands of mussels, and the high cost limits mussel research. Using genetic engineering to heterologously express mussel proteins is currently a popular research direction. Mfp-1 was the first identified protein, and attempts were made to recombinantly produce it. The decapeptide repeat sequence (6-20) of Mfp-1 is efficiently expressed in *E. coli*, with 6 decapeptide repeats expressed as inclusion bodies and 20 decapeptide repeats expressed in both soluble and insoluble forms. Mfp-3 is rich in Dopa, a key adhesion protein, and has 30-35 different variants. The cDNA sequences of Mgfp-3A and Mgfp-5 from *M. galloprovincialis* were identified. After codon optimization based on the codon usage preferences of *E. coli*, the yield of recombinant Mgfp-3A increased from 0.8 mg / L to 47 mg / L (half expressed as inclusion bodies); the yield of Mgfp-5 increased from 2.6 mg / L to 50 mg / L. In previous studies, our group achieved recombinant expression of another variant of *M. galloprovincialis*, 3B (Mgfp-3B), in *E. coli*, with a yield of ~51 mg / L after simple purification. However, Mgfp-3B expression is similar to Mgfp-3A, with half of the protein still being inclusion body protein.

[0004] Escherichia coli is the most commonly used host in the field of genetic engineering. It is simple to culture, produces cells quickly, and is easy to manipulate, thus it is widely used for expressing recombinant heterologous proteins. However, when expressing target proteins using recombinant DNA technology in E. coli, the lack of key elements for protein folding leads to low folding efficiency. Recombinant proteins easily aggregate to form biologically inactive inclusion bodies, requiring complex denaturation and renaturation processes to refold into functional proteins. Simultaneously, the expression level of the target protein produced by heterologous expression is also very low. Currently, several methods have been explored to address the problem of inclusion body expression. Fusion expression with tag proteins, co-expression with molecular chaperones, changing promoters or RBS sequences, and lowering the expression temperature are all attempts to solve these problems. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a bicistronic translation-coupled expression vector and further proposes a method for constructing genetically engineered bacteria that co-express mussel protein Mgfp-3B with molecular chaperones SUMO and / or TrxA.

[0006] The technical problem that this invention also aims to solve is to provide a fermentation medium and fermentation process for promoting the soluble expression of mussel protein Mgfp-3B.

[0007] In summary, this study aimed to explore a method for efficiently expressing soluble mussel proteins. While co-expression of molecular chaperones in *E. coli* can assist in the expression of exogenous genes, there is currently no method using molecular chaperones to enhance soluble expression in mussel protein research.

[0008] In this invention, the Mfp-3 variant B (Mgfp-3B) from *M. galloprovincialis* was recombinantly expressed in *E. coli*, with a molecular weight of only 12 kDa. Through genetic engineering, the molecular chaperone SUMO and / or TrxA genes were combined with the mussel protein Mgfp-3B on the expression vector pET-28a(+) to construct a bicistronic translation-coupled expression vector, which was then transformed into *E. coli* BL21(DE3) for expression. This achieved 60-70% of the target protein in a soluble form. Through continuous optimization of the fermentation medium and conditions, the yield of soluble mussel protein reached 200-300 mg / L.

[0009] To achieve the above objectives, the present invention provides a bicistronic translation-coupled expression vector, wherein a molecular chaperone, a novel ribosome binding site, and a target protein gene are inserted into the pET-28a(+) plasmid to construct a bicistronic translation-coupled expression vector, wherein the molecular chaperone gene is located upstream of the inserted novel ribosome binding site, and the mussel protein gene is located downstream of the inserted novel ribosome binding site, and the molecular chaperone is any one or a combination of two of SMUO and TrxA.

[0010] In one specific embodiment, the target protein gene is mussel protein Mgfp-3B, whose nucleotide sequence is shown in SEQ ID No: 1.

[0011] This invention further proposes a method for constructing the above-mentioned bicistronic translation-coupled expression vector, comprising the following steps:

[0012] (1) Cloning of the coding regions of mussel protein Mgfp-3B, molecular chaperone SUMO, TrxA and SUMO-TrxA genes, wherein an additional ribosome binding site was introduced in both the downstream primer of the molecular chaperone and the upstream primer of the mussel protein; when the mussel protein is linked to the molecular chaperone fragment, the newly introduced ribosome binding site acts as a linker peptide to avoid direct fusion expression of the two, and the bicistronic structure also avoids downstream molecular chaperone enzyme cleavage caused by fusion expression. The newly introduced ribosome binding site is independent of the ribosome binding site in the original plasmid and has a different sequence from the ribosome binding site in the original plasmid.

[0013] (2) The mussel protein Mgfp-3B was linked with molecular chaperones SUMO, TrxA and SUMO-TrxA respectively and transferred into plasmid pET-28a(+) to construct expression vectors pET-28a(+)-SUMO-Mgfp-3B, pET-28a(+)-TrxA-Mgfp-3B and pET-28a(+)-SUMO-TrxA-Mgfp-3B.

[0014] The nucleotide sequence of the encoding gene SUMO is shown in Sequence Listing 3, and its amino acid sequence is shown in Sequence Listing 4; the nucleotide sequence of the encoding gene TrxA is shown in Sequence Listing 5, and its amino acid sequence is shown in Sequence Listing 6.

[0015] The present invention further proposes a recombinant genetically engineered strain, which is obtained by transferring the above-mentioned bicistronic translation-coupled expression vector into the expression host E.coli BL21(DE3).

[0016] This invention further proposes the application of the above-mentioned bicistronic translation-coupled expression vector or recombinant genetically engineered strain in the preparation of soluble mussel protein.

[0017] Specifically, this invention proposes a method for preparing soluble mussel protein, which is a shake-flask fermentation method for preparing mussel protein, and specifically includes the following steps:

[0018] (1) The recombinant genetically engineered strain was activated on a plate and then inoculated into a shake flask containing liquid culture medium and cultured at 35-40℃ and 180-220rpm for 8-12h to be used as seed liquid.

[0019] (2) Inoculate the seed culture from step (1) into a shaker flask containing liquid culture medium at an inoculation rate of 1-10%, and incubate at 35-40℃ and 180-220 rpm. 600 When the concentration reaches 2-3, add an inducer with a final concentration of 0.1-1 mM, and continue culturing for 6-10 hours.

[0020] This invention also provides a method for preparing mussel protein by fermentation in a fermenter, comprising the following steps:

[0021] (1) The recombinant genetically engineered strain was activated on a plate and then inoculated into a shake flask containing liquid culture medium and cultured at 35-40℃ and 180-220rpm for 8-12h to be used as a primary seed liquid.

[0022] (2) Inoculate the primary seed culture from step (1) into a shaker flask containing liquid culture medium at an inoculation rate of 1-10%, and incubate at 35-40℃ and 180-220 rpm until OD. 600 Achieving a value of 5-6 allows for use as a secondary seed solution;

[0023] (3) The secondary seed liquid is inoculated into the fermenter at an inoculation rate of 1-10% for fermentation culture. The fermenter is filled with 40%-60% of the initial fermentation culture medium. The initial conditions are pH 7.0±0.5. During the fermentation process, the pH is automatically adjusted with 10-25% ammonia water and the temperature is 35-40℃. The dissolved oxygen is maintained at 20-40% by adjusting the rotation speed and aeration.

[0024] (4) After the initial carbon source is depleted, pH and DO will continue to rise. When pH is above 7.5 and dissolved oxygen exceeds 40%, fed culture medium should be added to maintain cell growth. During fermentation, OD should be measured every two hours. 600 When OD 600 At 50-70°C, IPTG is added to induce expression, with a final concentration of 0.1-1 mM. Induction ends when the cells grow slowly or the yield no longer increases.

[0025] Specifically, if the pH is above 7.5, the feeding rate should be increased to approximately 30 ml / h; if the pH is below 6.5, the feeding rate should be reduced to approximately 20 ml / h. To maintain cell growth, OD should be measured every two hours during fermentation. 600 When OD 600 At 50-70°C, IPTG is added to induce expression, with a final concentration of 0.1-1 mM. Induction ends when the cells grow slowly or the yield no longer increases.

[0026] The liquid culture medium is formulated as follows: 10-20 g / L peptone, 5-10 g / L yeast extract, 10-20 g / L sodium chloride, and 25-50 μg / ml kanamycin sulfate.

[0027] The initial fermentation medium formula is: yeast extract 10-40 g / L, peptone 10-40 g / L, glucose 5-20 g / L, ammonium sulfate 5-10 g / L, potassium dihydrogen phosphate 1-5 g / L, anhydrous magnesium sulfate 0.5-5 g / L, and kanamycin sulfate 5-50 μg / ml; the fed culture medium formula is: glycerol 400-600 g / L and yeast extract 100-400 g / L.

[0028] The feeding medium can be added in any of the following ways: one-time feeding, batch feeding, or fed-batch feeding.

[0029] Beneficial Effects: This invention establishes a simpler and more efficient method for expressing bioactive soluble mussel proteins, avoiding the cumbersome and inefficient refolding process required for inclusion body mussel proteins. Existing research often uses single molecular chaperones, sometimes with limited effectiveness. This study investigates the effects of single molecular chaperones SUMO and TrxA, and the combination of SUMO-TrxA, on soluble expression levels. Both SUMO and TrxA are small-molecule solubilizing tags, avoiding the weakening of target protein expression caused by excessively large molecular chaperones. Using individually inserted ribosome binding sites as linker peptides, the upstream molecular chaperone and downstream target protein work synergistically, achieving 60-70% soluble expression of mussel proteins. The molecular chaperones SUMO and TrxA increased soluble expression levels by 12.52% and 10.99%, respectively; while the combination of SUMO-TrxA increased it by 18.07%. Although numerous studies have been conducted using molecular chaperones to improve soluble expression, most have employed fusion expression to produce the target protein. Subsequent purification processes require the removal of molecular chaperones to prevent interference with the function of the target protein. However, the proteases required for chaperone removal are not only expensive but also lack specificity, resulting in low removal efficiency. Furthermore, excessive purification steps can reduce the recovery rate of the target protein. The bicistronic conjugated expression vector constructed in this study ensures the effective functioning of the molecular chaperone while avoiding downstream enzymatic cleavage operations associated with fusion expression, simplifying the process and reducing costs. The fermentation medium and fermentation process provided by this invention enable the OD of mussel protein Mgfp-3B to increase significantly. 600 With a concentration of 120-150, the yield of soluble mussel protein can reach 200-300 mg / L, which is several times higher than that of simple shake-flask fermentation in terms of both biomass and yield. The mussel protein prepared by this invention can be widely used in the fields of medicine and medical devices. Attached Figure Description

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0031] Figure 1 A framework diagram for constructing bicistronic translation-coupled expression vectors;

[0032] Figure 2 Cloning of the coding regions of mussel protein Mgfp-3B and molecular chaperones SUMO and TrxA genes; M: standard molecular weight of DNA; 1: PCR amplification product of Mgfp-3B in step 1 of Example 1, below 250bp; 2: PCR amplification product of Mgfp-3B in step 1 of Examples 2 and 3, below 250bp; 3: PCR amplification product of SUMO in step 2 of Example 1, approximately 300bp; 4: PCR amplification product of SUMO in step 4 of Example 3, approximately 300bp; 5: PCR amplification product of TrxA in step 2 of Example 2, above 300bp; 6: PCR amplification product of TrxA in step 2 of Example 3, above 300bp;

[0033] Figure 3 Linkage of mussel protein with molecular chaperone: M: standard molecular weight of DNA; 1: PCR amplification product of SUMO-TrxA-Mgfp-3B, below 900bp; 2: PCR amplification product of SUMO-Mgfp-3B, around 550bp; 3: PCR amplification product of TrxA-Mgfp-3B, below 600bp.

[0034] Figure 4 SDS-PAGE electrophoresis analysis of co-expressed strains; M: protein standard molecular weight;

[0035] SUMO-Mgfp-3B indicates co-expression of Mgfp-3B and SUMO; TrxA-Mgfp-3B indicates co-expression of Mgfp-3B and TrxA; SUMO-TrxA-Mgfp-3B indicates co-expression of Mgfp-3B and SUMO-TrxA; S indicates soluble expression, and IS indicates insoluble inclusion body expression. The molecular weight of Mgfp-3B is approximately 12 kDa.

[0036] Figure 5 For the expression level analysis of mussel protein in co-expressed strains, the soluble fraction represents the soluble expression yield, and the insoluble fraction represents the inclusion body expression yield.

[0037] Figure 6The growth curves of mussel protein Mgfp-3B fermented using the fermentation medium and fermentation process described in this experiment are shown in the figure. Detailed Implementation

[0038] In the following examples, the total protein concentration was determined using the Bradford Protein Assay Kit; the proportion of mussel protein to total protein was calculated using grayscale scanning with Gel-Pro Analyzer (Media Cybernatics, version 4.0.0.4); mussel protein yield was obtained by multiplying the target protein percentage by the total protein concentration; the proportion of soluble mussel protein to total mussel protein was calculated using grayscale scanning with Gel-Pro Analyzer (Media Cybernatics, version 4.0.0.4); and the soluble mussel protein yield was obtained by multiplying the soluble protein percentage by the total mussel protein concentration.

[0039] Example 1: Construction of a genetically engineered bacterium for producing soluble mussel protein Mgfp-3B.

[0040] 1. Obtaining the SUMO-Mgfp-3B gene fragment

[0041] (1) Design upstream and downstream primers based on the coding region sequence of Mgfp-3B. The upstream primer is designed from the start codon, and the downstream primer is designed up to the stop codon. A new ribosome binding site is introduced into the upstream primer (in italics).

[0042] Named RBS2, the designed primers are:

[0043] Upstream primer 3B-F1:

[0044] 5'-GGTGGTGGTAAGGAGGGCTAATGAACAACATCAGCGTTGC-3'

[0045] Downstream primer 3B-R:

[0046] 5'-TGGTGGTGGTGGTGCTCGAGATAGTTATATTTACGACGAC-3'

[0047] The PCR reaction system consisted of 25 μL: 2 μL Mgfp-3B genomic template DNA, 2 μL each of forward and reverse primers, 12.5 μL 2×PhantaMax Master Mix, and 6.5 μL ddH2O. The 2×PhantaMax Master Mix was purchased from Novizan (Nanjing, China). The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, followed by 30 cycles of 94℃ for 30 s, 60℃ for 30 s, and 72℃ for 15 s, with a final extension at 72℃ for 10 min. The PCR product was analyzed by 1% agarose gel electrophoresis, which showed a specific band below 250 bp. The Mgfp-3B gene fragment was recovered by gel excision.

[0048] (2) Based on the coding region sequence of SUMO, upstream and downstream primers were designed. The upstream primer was designed from the start codon, and the downstream primer was designed up to the stop codon. A new ribosome binding site was introduced into the downstream primer (in italics). The designed primers are:

[0049] Upstream primer SUMO-F:

[0050] 5'-GAAGGAGATATACCATGGGCATGAGCGATAGTGAAGTTAA-3'

[0051] Downstream primer SUMO-R1:

[0052] 5'-GTTCATTAGCCCTCCCTTACCACCACCAATCTGTTCACGAT-3'

[0053] The PCR reaction system and conditions were the same as in step 1(1). The PCR product was subjected to 1% agarose gel electrophoresis, and the results showed that there was a specific band at 300 bp. SUMO was recovered by gel excision.

[0054] (3) The SUMO and Mgfp-3B gene fragments were ligated by PCR using SUMO-F as the upstream primer and 3B-R as the downstream primer. The PCR reaction system was the same as in step 1(1). The PCR reaction conditions were: pre-denaturation at 94℃ for 5 min, followed by 30 cycles of 94℃ for 30 s, 60℃ for 30 s, and 72℃ for 20 s, with a final extension at 72℃ for 10 min. The PCR product was subjected to 1% agarose gel electrophoresis, and the results showed a specific band at 550 bp. The SUMO-Mgfp-3B gene fragment was recovered by gel excision.

[0055] 2. Obtaining the TrxA-Mgfp-3B gene fragment

[0056] (1) Based on the coding region sequence of Mgfp-3B, upstream and downstream primers were designed. The upstream primer was designed from the start codon, and the downstream primer was designed up to the stop codon. A new ribosome binding site (italicized) was introduced into the upstream primer and named RBS2. The designed primers are as follows:

[0057] Upstream primer 3B-F2:

[0058] 5'-CTGGCCGGTAAGGAGGGCTAATGAACAACATCAGCGTTGC-3'

[0059] Downstream primer 3B-R:

[0060] 5'-TGGTGGTGGTGGTGCTCGAGATAGTTATATTTACGACGAC-3'

[0061] The PCR reaction system and conditions were the same as in step 1(1). The PCR product was subjected to 1% agarose gel electrophoresis, and the results showed that there was a specific band below 250 bp. The Mgfp-3B gene fragment was recovered by gel excision.

[0062] (2) Based on the coding region sequence of TrxA, upstream and downstream primers were designed. The upstream primer was designed from the start codon, and the downstream primer was designed up to the stop codon. A new ribosome binding site, RBS2, was introduced into the downstream primer. The designed primers are as follows:

[0063] Upstream primer TrxA-F1:

[0064] 5'-GAAGGAGATATACCATGGGCATGAGCGATAAAATCATCCA-3'

[0065] Downstream primer TrxA-R:

[0066] 5'-GTTCATTAGCCCTCCTTACCGGCCAGATTGGCATCCAGAA-3'

[0067] The PCR reaction system and conditions were the same as in step 1(1). The PCR product was subjected to 1% agarose gel electrophoresis, and the results showed that there was a specific band above 300 bp. TrxA was recovered by gel excision.

[0068] (3) The gene fragments TrxA and Mgfp-3B were ligated by PCR, with the upstream primer TrxA-F1 and the downstream primer 3B-R. The PCR reaction system and conditions were the same as in step 1(3). The PCR product was gelled on a 1% agarose gel, and the results showed that there was a specific band below 600 bp. The TrxA-Mgfp-3B gene fragment was recovered by gel excision.

[0069] 3. Obtaining the SUMO-TrxA-Mgfp-3B gene fragment

[0070] (1) Obtaining the Mgfp-3B gene fragment is the same as in step 2(1).

[0071] (2) Based on the coding region sequence of TrxA, upstream and downstream primers were designed. The upstream primer was designed from the start codon, and the downstream primer was designed up to the stop codon. A new ribosome binding site, RBS2, was introduced into the downstream primer. The designed primers are as follows:

[0072] Upstream primer TrxA-F2:

[0073] 5'-ATCGTGAACAGATTGGTGGTATGAGCGATAAAATCATCCA-3'

[0074] Downstream primer TrxA-R:

[0075] 5'-GTTCATTAGCCCTCCTTACCGGCCAGATTGGCATCCAGAA-3'

[0076] The PCR reaction system and conditions were the same as in step 1(1). The PCR product was subjected to 1% agarose gel electrophoresis, and the results showed that there was a specific band above 300 bp. TrxA was recovered by gel excision.

[0077] (3) The gene fragments TrxA and Mgfp-3B were ligated by PCR, with the upstream primer TrxA-F2 and the downstream primer 3B-R. The PCR reaction system and conditions were the same as in step 1(3). The PCR product was gelled on a 1% agarose gel, and the results showed that there was a specific band below 600 bp. The TrxA-Mgfp-3B gene fragment was recovered by gel excision.

[0078] (4) Design upstream and downstream primers based on the coding region sequence of SUMO. The upstream primer is designed from the start codon, and the downstream primer is designed up to the stop codon. The designed primers are:

[0079] Upstream primer SUMO-F:

[0080] 5'-GAAGGAGATATACCATGGGCATGAGCGATAGTGAAGTTAA-3'

[0081] Downstream primer SUMO-R2:

[0082] 5'-TGGATGATTTTATCGCTCATACCACCAATCTGTTCACGAT-3'

[0083] The PCR reaction system and conditions were the same as in step 1(1). The PCR product was subjected to 1% agarose gel electrophoresis, and the results showed that there was a specific band at 300 bp. SUMO was recovered by gel excision.

[0084] (5) The SMUO and TrxA-Mgfp-3B gene fragments were ligated by PCR using SUMO-F as the upstream primer and 3B-R as the downstream primer. The PCR reaction system was the same as in step 1(3). The PCR reaction conditions were: pre-denaturation at 94℃ for 5 min, followed by 30 cycles of 94℃ for 30 s, 60℃ for 30 s, and 72℃ for 25 s, with a final extension at 72℃ for 10 min. The PCR product was subjected to 1% agarose gel electrophoresis, and the results showed a specific band below 900 bp. The SMUO-TrxA-Mgfp-3B gene fragment was recovered by gel excision.

[0085] 4. Construction of Bicistronic Translation-Coupled Expression Vectors

[0086] (1) A small amount of pET-28a empty plasmid was extracted. The PCR-recovered products (SUMO-Mgfp-3B, TrxA-Mgfp-3B, SUMO-TrxA-Mgfp-3B) and plasmid pET-28a were double-digested with restriction endonucleases Nco I and Xho I. The digestion products were purified by 1% agarose gel and then recovered for later use.

[0087] (2) The enzyme digestion products SUMO-Mgfp-3B, TrxA-Mgfp-3B, and SUMO-TrxA-Mgfp-3B were ligated to the linearized pET-28a plasmid. The reaction system consisted of 5 μL of linearized vector, 3 μL of insert fragment, 2 μL of Exnase II, 4 μL of 5×CE II Buffer, and 6 μL of ddH2O. Exnase II and 5×CE II Buffer were purchased from Novizan (Nanjing, China). After reacting 20 μL of the reaction system on ice for 30 minutes, 100 μL of E. coli DH5α competent bacterial culture was added and the mixture was kept on ice for another 30 minutes. The mixture was then heat-shocked at 42℃ for 60–90 seconds, followed by recovery culture with 5 times the volume of LB liquid medium (without antibiotics) for 1 hour. Afterward, an appropriate volume was plated (with kanamycin resistance) and incubated at 37℃ for 12 hours. Single clones were screened, cultured, and plasmids were extracted and sequenced to obtain the corresponding bicistronic expression vectors pET-28a(+)-SUMO-Mgfp-3B, pET-28a(+)-TrxA-Mgfp-3B, and pET-28a(+)-SUMO-TrxA-Mgfp-3B. The recombinant plasmids were then transformed into *E. coli* BL21(DE3) for expression.

[0088] Example 2: Preparation of soluble mussel protein by shake-flask fermentation.

[0089] The three recombinant genetically engineered bacteria strains SUMO-Mgfp-3B, TrxA-Mgfp-3B, and SUMO-TrxA-Mgfp-3B successfully constructed in Example 1 were activated by streaking on agar plates. Single colonies grown on the plates were picked and inoculated into shake flasks containing 5 ml of liquid culture medium and cultured overnight at 37°C with shaking at 200 rpm for 10 h. A 10% inoculum was then transferred to a 1 L baffled shake flask containing 200 mL of liquid culture medium and cultured at 37°C with 200 rpm until the OD600 reached 2–3. IPTG was then added to a final concentration of 1 mM / L, and cultured for another 6 h at 37°C with 200 rpm. After culturing, the bacterial culture was centrifuged and the expression of the recombinant protein was determined by SDS-PAGE electrophoresis. The expression results are shown in Table 1 below.

[0090] Table 1

[0091]

[0092] In Example 2, the apparent molecular weight of Mgfp-3B generated after induction by SUMO-Mgfp-3B, TrxA-Mgfp-3B, and SUMO-TrxA-Mgfp-3B constructed in this study was approximately 12 kDa on SDS-PAGE gel, consistent with the relative molecular mass of Mgfp-3B expressed alone, indicating successful heterologous expression of mussel protein. Only 43.8% of the target protein in the Mgfp-3B expressed alone was in a soluble form; however, when co-expressed with molecular chaperones SUMO, TrxA, and SUMO-TrxA, the soluble proportion of the target protein was increased, reaching 56.32%, 54.79%, and 61.87%, respectively. Compared to SUMO (12.52%) and TrxA (10.99%), SUMO-TrxA showed the best improvement, increasing the solubility by 18.07%, demonstrating that the molecular chaperones selected in this invention can effectively improve the solubility of the target protein, and that the combined effect of molecular chaperones is far superior to that of a single molecular chaperone.

[0093] Example 3: Preparation of soluble mussel protein by fermentation in a fermenter.

[0094] (1) The genetically engineered bacterium SUMO-TrxA-Mgfp-3B that produces soluble mussel protein was activated on a plate and then inoculated into a shake flask containing 5 ml of liquid culture medium. It was cultured at 37°C and 200 rpm for 10 h to be used as a primary seed culture.

[0095] (2) Inoculate the primary seed culture from step (1) at a rate of 10% into a shaker flask containing 200 ml of liquid culture medium, and incubate at 37°C and 200 rpm until OD. 600 A solution with a value of 5-6 can be used as a secondary seed solution.

[0096] (3) The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 10% for fermentation culture. The fermenter contained 2L of initial fermentation medium with initial conditions of pH 7.0±0.5. During fermentation, the pH was automatically adjusted with 25% ammonia water, the temperature was 37℃, and the dissolved oxygen was maintained at 20% by adjusting the rotation speed and aeration.

[0097] (4) Once the initial carbon source is depleted, pH and DO will continue to rise, exceeding the set values. At this point, fed culture medium should be added to maintain cell growth. The feeding rate should be maintained at 20-30 ml / h, adjusted according to pH. If the pH is above 7.5, the feeding rate should be increased to approximately 30 ml / h; if the pH is below 6.5, the feeding rate should be reduced to approximately 20 ml / h. OD should be measured every two hours during fermentation. 600 When OD 600 At 60°C, IPTG was added to induce expression, with a final concentration of 0.2 mM. Induction for this batch lasted 12 hours.

[0098] (5) The fermentation broth is centrifuged to collect the cells, and the supernatant is collected after the cells are broken for subsequent purification.

[0099] (6) The supernatant and precipitate of the lysate were subjected to SDS-PAGE electrophoresis to examine the fermentation expression level and the expression level of soluble proteins.

[0100] The liquid culture medium formula is as follows: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride and 25 μg / ml kanamycin sulfate.

[0101] The initial fermentation medium formula was: 20 g / L yeast extract, 10 g / L peptone, 15 g / L glucose, 5 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 1 g / L anhydrous magnesium sulfate and 25 μg / ml kanamycin sulfate; the fed medium formula was: 400 g / L glycerol and 200 g / L yeast extract.

[0102] Example 4: Preparation of soluble mussel protein by fermentation in a fermenter (as a control example).

[0103] (1)(2)(3) Same as Example 2.

[0104] (4) Once the initial carbon source is depleted, pH and DO will continue to rise, exceeding the set values. At this point, fed culture medium should be added to maintain cell growth. The feeding rate should be maintained at 20-30 ml / h, adjusted according to pH. If the pH is above 7.5, the feeding rate should be increased to approximately 30 ml / h; if the pH is below 6.5, the feeding rate should be reduced to approximately 20 ml / h. OD should be measured every two hours during fermentation. 600 When OD600 At 40°C, IPTG was added to induce expression, with a final concentration of 0.2 mM. Induction for this batch lasted 12 hours.

[0105] The feed culture medium formula involved in step 4 is: glucose 600g / L.

[0106] (5) Centrifuge the fermentation broth to collect the cells, break the cells and collect the supernatant for subsequent purification.

[0107] (6) The supernatant and precipitate of the lysate were subjected to SDS-PAGE electrophoresis to examine the fermentation expression level and the expression level of soluble proteins.

[0108] The experimental results are shown in the table below:

[0109]

[0110] Among them, the highest OD600 is the highest biomass during the entire fermentation process.

[0111] The results above show that, due to the use of the fermentation medium formulation and process of this invention, Examples 3 and 4 of this invention achieved 60-70% soluble expression of the target protein. The soluble expression level in Example 3 reached 224.35 mg / L; the soluble expression level of the target protein in Example 4 was significantly lower than that in Example 3.

[0112] This invention provides a method and approach for promoting the soluble expression of recombinant mussel protein Mgfp-3B. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A bicistronic translation-coupled expression vector, characterized in that, The expression vector is inserted with a molecular chaperone, a new ribosome binding site and a target protein gene on a pET-28a(+) plasmid to construct a bicistronic translation coupling expression vector, wherein the molecular chaperone gene is located upstream of the inserted new ribosome binding site, and the target protein gene is located downstream of the inserted new ribosome binding site, the target protein gene is a mussel protein gene, and the molecular chaperone is a combination of SUMO and TrxA; the mussel protein gene is Mgfp-3B, the nucleotide sequence of which is shown as SEQ ID No: 1; the nucleotide sequence of the gene SUMO is shown as SEQ ID No: 3, and the amino acid sequence is shown as SEQ ID No: 4; the nucleotide sequence of the gene TrxA is shown as SEQ ID No: 5, and the amino acid sequence is shown as SEQ ID No: 6; wherein the construction method of the bicistronic translation coupling expression vector comprises the following steps: (1) Cloning of the mussel protein gene and the molecular chaperone SUMO-TrxA gene coding region, wherein an additional ribosome binding site is introduced into the downstream primer of the molecular chaperone and the upstream primer of the mussel protein: The upstream and downstream primers are designed according to the coding region sequence of Mgfp-3B, the upstream primer is designed from the start codon, and the downstream primer is designed to the stop codon, wherein the primer with a new ribosome binding site introduced in the upstream primer is: Upstream primer 3B-F2: 5'-CTGGCCGG TAAGGAG GGCTAATGAACAACATCAGCGTTGC-3'; The downstream primer 3B-R is 5'-TGGTGGTGGTGGTGCTCGAGATAGTTATATTTACGACGAC-3'; The PCR product is subjected to 1% agarose gel electrophoresis, and the Mgfp-3B gene fragment is recovered by cutting the gel; The upstream and downstream primers are designed according to the coding region sequence of TrxA, the upstream primer is designed from the start codon, and the downstream primer is designed to the stop codon, wherein a new ribosome binding site is introduced into the downstream primer; the designed primer is: The upstream primer TrxA-F2 is 5'-ATCGTGAACAGATTGGTGGTATGAGCGATAAAATCATCCA-3'; Downstream primer TrxA-R: 5'- GTTCATTAGC CCTCCTTA CCGGCCAGATTGGCATCCAGAA-3'; The PCR product is subjected to 1% agarose gel electrophoresis, and the gene fragment of TrxA is recovered by cutting the gel; The gene fragments of TrxA and Mgfp-3B are connected by PCR, and the PCR product is subjected to 1% agarose gel electrophoresis to obtain the TrxA-Mgfp-3B gene fragment by cutting the gel; The upstream and downstream primers are designed according to the coding region sequence of SUMO, the upstream primer is designed from the start codon, and the downstream primer is designed to the stop codon, and the designed primer is: The upstream primer SUMO-F is 5'-GAAGGAGATATACCATGGGCATGAGCGATAGTGAAGTTAA-3'; The downstream primer SUMO-R2 is 5'-TGGATGATTTTATCGCTCATACCACCAATCTGTTCACGAT-3'; The PCR product is subjected to 1% agarose gel electrophoresis to obtain the SUMO fragment by cutting the gel; The gene fragment of SMUO and TrxA-Mgfp-3B is connected by PCR, and the PCR product is recovered by 1% agarose gel to obtain the gene fragment of SMUO-TrxA-Mgfp-3B; The SMUO-TrxA-Mgfp-3B gene fragment was transferred into plasmid pET-28a(+) to construct an expression vector pET-28a(+) SUMO-TrxA-Mgfp-3B .

2. A recombinant genetically engineered bacterial strain, characterized in that, The recombinant genetically engineered strain is obtained by transforming the bi- cistronic translation coupling expression vector of claim 1 into an expression host E. coli BL21(DE3).

3. The use of the bicistronic translation coupling expression vector of claim 1 or the recombinant genetically engineered strain of claim 2 in the preparation of soluble mussel protein.

4. A method of preparing soluble mussel protein, characterized by, The method comprises the following steps: (1) the recombinant genetically engineered strain of claim 2 is activated by plate and inoculated into a shake flask containing a liquid culture medium, and cultured at 35-40℃ and 180-220rpm for 8-12h to obtain a seed liquid; (2) The seed liquid in step (1) is inoculated into a baffle shake flask containing liquid medium at an inoculation amount of 1-10%, and cultured at 35-40°C and 180-220 rpm. When the OD 600 When the OD reaches 2-3, an inducer is added at a final concentration of 0.1-1 mM, and the culture is continued for 6-10 h to end.

5. A method of preparing soluble mussel protein, characterized by, The method comprises the following steps: (1) the recombinant genetically engineered strain of claim 2 is activated by plate and inoculated into a shake flask containing a liquid culture medium, and cultured at 35-40℃ and 180-220rpm for 8-12h to obtain a seed liquid; (2) The primary seed liquid in step (1) is inoculated into a baffle shake flask containing liquid medium at an inoculation amount of 1-10%, and cultured at 35-40°C and 180-220 rpm until OD 600 5-6 is reached and used as secondary seed liquid; (3) the secondary seed liquid is inoculated into a fermenter at an inoculation amount of 1-10% for fermentation culture, the fermenter contains 40-60% of the initial fermentation medium with a volume of the fermenter, the initial conditions are pH 7.0±0.5, and the pH is automatically adjusted to 7.0±0.5 by using ammonia with a mass concentration of 10-25% during the fermentation process; the temperature is 35-40℃; and the dissolved oxygen is maintained at 20-40% by adjusting the rotation speed and aeration. (4) When the initial carbon source is exhausted, the pH and DO continue to rise, when the pH is higher than 7.5 and the dissolved oxygen is more than 40%, at this time, the feed medium is added to maintain the growth of the bacteria, and the OD is measured every two hours during the fermentation process 600 When the OD 600 At 50-70, the inducer IPTG is added to induce expression, and the final concentration of the inducer is 0.1-1 mM. When the bacteria grow slowly or the yield no longer increases, the induction is completed.

6. The production method according to claim 4 or 5, characterized by, The liquid culture medium formula is: 10-20 g / L peptone, 5-10 g / L yeast powder, 10-20 g / L sodium chloride, and 25-50 μg / ml kanamycin sulfate.

7. The preparation method according to claim 5, characterized in that, The initial fermentation medium formula is: yeast powder 10-40 g / L, peptone 10-40 g / L, glucose 5-20 g / L, ammonium sulfate 5-10 g / L, potassium dihydrogen phosphate 1-5 g / L, anhydrous magnesium sulfate 0.5-5 g / L, and 5-50 μg / mL kanamycin sulfate; and the feeding medium formula is: glycerol 400-600 g / L and yeast powder 100-400 g / L.

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