Method for improving protein yield and application

Abstract

The invention discloses a functional peptide for improving the yield of recombinant protein. The sequence of the functional peptide is shown as any one of SEQ ID No.1-15. The invention also discloses a coding gene and application thereof, and a corresponding method for improving the protein yield. The functional peptide can obviously improve the yield of recombinant protein, can obviously reduce the loss rate of the recombinant protein in an MtudeltaI-CM protein expression system in the expression and purification process, reduces the production cost of the recombinant protein, improves the stability of target protein, is suitable for production of various recombinant human-derived proteins, and has wide application prospects. The method lays a foundation for large-scale and low-cost mass production of various recombinant proteins, and has a good commercial application prospect.

Description

Technical Field

[0001] The invention relates to a method for improving protein yield and application thereof, belonging to the technical field of protein expression. Background Art

[0002] Recombinant proteins play a vital role in biomedicine, cosmetics and food industries. Due to the growing market demand, the use of engineered microorganisms to produce recombinant proteins on a large scale and efficiently has aroused great interest. To this end, various expression systems have been developed, including prokaryotes, yeast, mammalian and human cells, plants and insects. Among them, Escherichia coli remains the most widely used system due to its rapid growth, versatility and well-characterized genetic background. Although several recombinant protein bioproduction processes have been commercially successful, they still face challenges such as high costs and multi-step column purification required in downstream processing. Therefore, how to achieve efficient and large-scale expression of recombinant proteins, how to reduce separation costs and improve recovery efficiency are important research topics in industrial biotechnology (J.Kaur et al., International Journal of Biological Macromolecules, 2018, 106: 803-822).

[0003] Although protein purification technology is now mature and widely used, there are still many challenges, such as low recovery rate and poor stability of recombinant proteins. Usually, after the recombinant protein is synthesized by microorganisms, insoluble cell residues are removed mainly by centrifugation or filtration, and the supernatant containing the recombinant protein is purified by several steps of chromatographic columns such as affinity protein A chromatography, cation exchange chromatography and anion exchange chromatography, and the virus is removed and inactivated. After purification, the recombinant protein is replaced into an appropriate buffer by ultrafiltration or diafiltration, and stored in the form of a stock solution or added to the final preparation components in the form of a semi-finished product. With the increase of these purification steps, the recovery rate of the recombinant protein will gradually decrease. In addition, during the entire production process, the protein undergoes a variety of destructive factors, such as low pH, high salt, freeze-thaw, light, oscillation, shear, and various (hydrophobic) surfaces, all of which may cause structural changes or degradation of the protein and affect the stability of the protein.

[0004] Column-free protein purification methods are currently considered to be an effective alternative to traditional chromatography techniques. Column-free purification methods use polymeric tags to promote the selective aggregation of target proteins during or after expression in host cells and recover them from crude extracts by simple centrifugation. Currently, typical polymeric tags mainly consist of two parts; first, an aggregation-prone sequence and second, a cleavable site. The cleavable site is the removal of the aggregation-prone sequence by chemical methods, protease-mediated methods or intein-mediated cleavage. Recently, a cleavable self-aggregation tag scheme has been developed for column-free purification of recombinant proteins, achieving high yield and purity of the target recombinant protein. The self-aggregation tag scheme includes self-assembling peptides, PT linkers, pH-induced intron MtuΔI-CM and target proteins. The fusion protein is expressed in the host cells as insoluble aggregates, and the target protein is released into the supernatant in vitro by pH changes, thereby purifying the target protein. At present, this self-aggregation tag scheme has been successfully applied to the purification of recombinant proteins and peptides, such as human growth hormone, interferon α2a and brain natriuretic peptide. However, this approach faces challenges, mainly including premature cleavage of the fusion protein in the cell, resulting in a significant decrease in yield (Z. Lin et al., Aiche Journal, 2020, 66(3): e16806).

[0005] Studies have shown that although site mutations (H73Y / T430V, H73V / T430S or H73V / T430C) of MtuΔI-CM have been shown to reduce the premature cleavage rate of fusion proteins from 87% to 27-45% when using this protocol for lipase A production, the cleavage activity of MtuΔI-CM is regulated by the carboxyl-terminal flanking residues (C+1 or C+2). When this protocol is used for the production of other proteins, the fusion protein still undergoes severe premature cleavage. When the N-terminal residue of the target protein is cysteine ​​(Cys), serine (Ser) or histidine (His), the premature cleavage rate of the fusion protein in this protocol is as high as 79%. When using this system to produce different recombinant proteins, each recombinant protein needs to be modified differently, which not only increases the complexity of the operation but also increases the production cost.

[0006] Therefore, there is still a need in the art to develop more effective recombinant protein expression and purification strategies for use in low-cost, simple, and efficient recombinant protein production. Summary of the invention

[0007] The purpose of the present invention is to provide a functional peptide for improving the yield of recombinant protein.

[0008] The technical solution adopted by the present invention is:

[0009] A functional peptide for improving the yield of a recombinant protein, wherein the sequence of the functional peptides shown in any one of SEQ ID No.1-15 is formed by combining two of the functional peptides shown in SEQ ID No.1 to SEQ ID No.8.

[0010] The base sequence of the gene encoding the aforementioned functional peptide is shown in any one of SEQ ID No.19-33.

[0011] The application of the aforementioned functional peptides in assisting the expression of target proteins.

[0012] An expression vector, wherein the vector comprises an expression gene for a recombinant protein, wherein the recombinant protein is formed by adding the aforementioned functional peptide in front of a target protein, and the N-terminus of the target protein is connected to the C-terminus of the functional peptide.

[0013] The expression host cell is formed by transforming the aforementioned expression vector into a host cell, and is used to express a recombinant protein composed of a functional peptide and a target protein.

[0014] A method for increasing protein production comprises fusing the aforementioned functional peptide with a target protein for expression.

[0015] The aforementioned functional peptide is used in assisting the MtuΔI-CM protein expression system to express the target protein. The MtuΔI-CM protein expression system comprises a self-aggregating peptide L6KD, a PT-type linker and a pH-induced self-assembling peptide MtuΔI-CM; the N-terminus of the recombinant protein is connected to the C-terminus of the pH-induced self-assembling peptide MtuΔI-CM through the functional peptide in the functional peptide library to form a fusion protein.

[0016] MtuΔI-CM expression vector, inserting the aforementioned functional peptide between the target protein and the intein.

[0017] The MtuΔI-CM expression host cell is obtained by transforming the aforementioned MtuΔI-CM expression vector into a host cell, and is used to express a recombinant protein composed of a functional peptide and a target protein.

[0018] The host cell is selected from prokaryotes, yeasts and higher eukaryotic cells, wherein the prokaryotes include bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas and Streptomyces, preferably Escherichia coli.

[0019] The application of the aforementioned functional peptides in improving protein stability.

[0020] Beneficial effects of the present invention:

[0021] The functional peptide of the present invention can play a role in two different expression systems, not only can it significantly increase the yield of recombinant proteins, but also can significantly reduce the loss rate of recombinant proteins in the MtuΔI-CM protein expression system during expression and purification, reduce the production cost of recombinant proteins, and improve the stability of target proteins. It is suitable for the production of various recombinant human proteins, lays a foundation for large-scale, low-cost mass production of various recombinant proteins, and has good commercial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the structure of the protein expression system containing the functional peptide library and the expression vector map (pET30a expression system).

[0023] Figure 2 Schematic diagram of the structure of the protein expression system containing the functional peptide library and the expression vector map (MtuΔI-CM expression system).

[0024] Figure 3 Agarose gel electrophoresis diagram of the construction of protein expression system containing functional peptide library. M: DL5000 marker, the upper figure is pET30a expression system: 1: L0-C (control group without linker), 2: L1-C, 3: L2-C, 4: L3-C, 5: L4-C, 6: L5-C, 7: L6-C, 8: L7-C, 9: L8-C, 10: L9-C, 11: L10-C, 12: L11-C, 13: L12-C, 14: L13-C, 15: L14-C, 16: L15-C; the lower figure is MtuΔI- CM expression system: 1: M-L0-C (control group without linker), 2: M-L1-C, 3: M-L2-C, 4: M-L3-C, 5: M-L4-C, 6: M-L5-C, 7: M-L6-C, 8: M-L7-C, 9: M-L8-C, 10: M-L9-C, 11: M-L10-C, 12: M-L11-C, 13: M-L12-C, 14: M-L13-C, 15: M-L14-C, 16: M-L15-C.

[0025] Figure 4SDS-PAGE diagram of recombinant proteins expressed based on the protein expression system containing the functional peptide library. The upper figure is the pET30a expression system: 1: L0-C (control group without linker), 2: L1-C, 3: L2-C, 4: L3-C, 5: L4-C, 6: L5-C, 7: L6-C, 8: L7-C, 9: L8-C, 10: L9-C, 11: L10-C, 12: L11-C, 13: L12-C, 14: L13-C, 15: L14-C, 16: L15-C; the lower figure is the MtuΔI-CM expression system: 1 : M-L0-C (control group without linker), 2: M-L1-C, 3: M-L2-C, 4: M-L3-C, 5: M-L4-C, 6: M-L5-C, 7: M-L6-C, 8: M-L7-C, 9: M-L8-C, 10: M-L9-C, 11: M-L10-C, 12: M-L11-C, 13: M-L12-C, 14: M-L13-C, 15: M-L14-C, 16: M-L15-C. A: type III collagen (COL-III), B: fibronectin (FN), C: fusion protein (FP).

[0026] Figure 5 SDS-PAGE of the recombinant protein COL-III containing functional peptides after enrichment and purification. M: protein marker, 1: L12-C (MtuΔI-CM expression system), 2: L0-C (MtuΔI-CM expression system), 3: L0-C (pET30a expression system), 4: L12-C (pET30a expression system), 5: 0.5 mg / ml bovine serum albumin.

[0027] Figure 6SDS-PAGE image of the stability test of the recombinant protein after purification based on the protein expression system containing the functional peptide library. M: protein marker, 1: COL-III sample containing functional peptide L3 after treatment (placed at room temperature for 60 days, pET30a expression system), 2: COL-III sample containing functional peptide L10 after treatment (placed at room temperature for 60 days, pET30a expression system), 3: COL-III sample containing functional peptide L12 after treatment (placed at room temperature for 60 days, pET30a expression system), 4: COL-III sample containing functional peptide L15 after treatment (placed at room temperature for 60 days, pET30a expression system), 5: COL-III sample without functional peptide after treatment (placed at room temperature for 60 days, control) , 6: The treated COL-III sample without functional peptide (placed at room temperature for 60 days, control), 7: The treated COL-III sample containing functional peptide L3 (placed at room temperature for 60 days, MtuΔI-CM expression system), 8: The treated COL-III sample containing functional peptide L10 (placed at room temperature for 60 days, MtuΔI-CM expression system), 9: The treated COL-III sample containing functional peptide L12 (placed at room temperature for 60 days, MtuΔI-CM expression system), 10: The treated COL-III sample containing functional peptide L15 (placed at room temperature for 60 days, MtuΔI-CM expression system).

[0028] Figure 7 The fusion protein (FP) containing functional peptide emits green fluorescence under UV irradiation. 1: L0-FP (pET30a, control), 2: L3-FP (MtuΔI-CM), 3: L10-FP (MtuΔI-CM), 4: L12-FP (MtuΔI-CM). DETAILED DESCRIPTION

[0029] The present invention is further illustrated by examples below, but is not intended to be limiting of the present invention. The specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, and materials identical or similar to the types, models, qualities, properties or functions of the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0030] Example 1: Preparation of protein expression system plasmid containing functional peptide library

[0031] (1) Gene design and synthesis

[0032] In the present invention, a recombinant protein expression vector containing functional peptides L1-L15 in the functional peptide library is constructed, and human type III collagen (COL-III), fibronectin (FN) and a fusion protein (FP) formed by connecting fibronectin and green fluorescent protein through PTlinker are selected as the target proteins for the test, and their corresponding amino acid sequences are shown in SEQ ID No.16-18; the amino acid sequences of functional peptides L1-L15 in the functional peptide library are shown in SEQ ID No.1 to SEQ ID No.15, and the corresponding coding gene sequences are shown in SEQ ID No.19 to SEQ ID No.33. The functional peptides L1-L15, the corresponding coding nucleotide sequences of the recombinant proteins COL-III, FN, and FP, and the Gibson assembly primers are synthesized by Bio-Industry.

[0033] (2) Construction of recombinant expression vector

[0034] The above synthesized gene fragment was used as a template for PCR amplification. ① pET30a expression system: The target gene fragment containing the functional peptide in the functional peptide library and the pET30a expression vector backbone were connected by Gibson assembly to obtain the corresponding plasmid, wherein the primer sequences are shown in Table 1 and the plasmid map is shown in Table 1. Figure 1 ②MtuΔI-CM expression system: The MtuΔI-CM expression framework, the target gene fragment containing the functional peptide in the functional peptide library and the pET30a expression vector backbone were connected by Gibson assembly to obtain the corresponding plasmid, wherein the primer sequences are shown in Table 2 and the plasmid map is shown in Figure 2 As shown, L0 refers to the control, i.e., the target protein without the addition of functional peptide.

[0035] Table 1 Primer sequences of pET30a expression system

[0036]

[0037]

[0038] Table 2 Primer sequences of MtuΔI-CM expression system

[0039]

[0040]

[0041] Table 3 PCR amplification system

[0042]

[0043]

[0044] After preparing the PCR system shown in Table 3, mix and centrifuge. The PCR amplification conditions are as follows: the first stage is 98°C pre-denaturation for 30s; the second stage is 98°C denaturation for 10s, 50-72°C annealing for 30s, 72°C extension for 30s / kb, 32 cycles; the third stage is 72°C final extension for 2min. The above vector and gene fragment were recovered using a universal DNA purification kit (Tiangen Biochemical Technology Co., Ltd.) and the operating steps in the product manual were followed.

[0045] Table 4 Gibson connection system

[0046] System components Ingredient volume Gibson Assembly Master Mix(2X) 5μL Connect fragments 0.2–1pmols*XμL <![CDATA[dd H 2 The]]> (5-X)μL Overall system 10μL

[0047] After mixing the above components on ice, place in a 37°C heat bath for 60 min. The obtained ligation product is stored on ice or at -20°C for subsequent competent cell transformation.

[0048] The ligation product was transformed into the host bacterium E. coli DH5α by heat shock method, spread on LB culture resistance plate, cultured in a 37℃ constant temperature incubator overnight, and single clones were picked for colony PCR. The results were as follows: Figure 3As shown. The positive clones obtained initially were transferred to LB liquid culture medium and cultured overnight in a shaker at 37°C and 220rpm. The plasmids were extracted using a plasmid rapid extraction kit, and the successfully constructed plasmids were named pET30a expression system: L0-C, L1-C (functional peptide L1-COL-III), L2-C, L3-C, L4-C, L5-C, L6-C, L7-C, L8-C, L9-C, L10-C, L11-C, L12-C, L13-C, L14-C, L15-C; L0-F, L1-F ( Functional peptides L1-FN), L2-F, L3-F, L4-F, L5-F, L6-F, L7-F, L8-F, L9-F, L10-F, L11-F, L12-F, L13-F, L14-F, L15-F; L0-P, L1-P (functional peptide L1-FP), L2-P, L3-P, L4-P, L5-P, L6-P, L7-P, L8-P, L9-P, L10-P, L11-P, L12-P, L13-P, L14-P, L15-P; MtuΔI-CM table Da system: M-L0-C, M-L1-C (MtuΔI-CM-functional peptide L1-COL-III), M-L2-C, M-L3-C, M-L4-C, M-L5-C, M-L6-C, M-L7-C, M-L8-C, M-L9-C, M-L10-C, M-L11-C, M-L12-C, M-L13-C, M-L14-C, M-L15-C; M-L0-F, M-L1-F, M-L2-F, M-L3-F, M-L4-F, M-L5-F, M-L 6-F, M-L7-F, M-L8-F, M-L9-F, M-L10-F, M-L11-F, M-L12-F, M-L13-F, M-L14-F, M-L15-F; M-L0-P, M-L1-P, M-L2-P, M -L3-P, M-L4-P, M-L5-P, M-L6-P, M-L7-P, M-L8-P, M-L9-P, M-L10-P, M-L11-P, M-L12-P, M-L13-P, M-L14-P, M-L15-P.

[0049] (3) Construction of engineered bacteria

[0050] The recombinant expression plasmid obtained above was transferred into Escherichia coli BL21 (DE3) competent cells by chemical transformation, and positive engineering bacteria were obtained by antibiotic plate screening. The specific process was as follows: ① 4 μL of the recombinant expression plasmid was added to 100 μL of Escherichia coli competent cells BL21 (DE3) and placed on ice for 30 min; ② the mixture was heat-shocked in a 42°C water bath for 90 s, and then quickly placed on ice for 2 min; ③ 900 μL of LB liquid medium without resistance (10 g / L peptone, 5 g / L PBS) was added to the mixture. / L yeast extract, 10g / L sodium chloride), and cultured at 37°C, 220rpm in a shaker for 1h; ④ Take 200μL of the bacterial solution and evenly spread it on an LB solid culture medium plate containing ampicillin (10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, 15g / L agar, 100μg / mL ampicillin); ⑤ Incubate the plate upside down in a 37°C incubator for about 16h, and wait for clearly visible colonies to grow, and obtain the corresponding engineering strain pET30a expression system: C0, C1, C 2. C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18; F0, F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14, F15; P0, P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12, P13, P14, P15; MtuΔI-CM expression system: M0, M1, M 2. M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, M13, M14, M15; MF0, MF1, MF2, MF3, MF4, MF5, MF6, MF7, MF8, MF9, MF10, M F11, MF12, MF13, MF14, MF15; MP0, MP1, MP2, MP3, MP4, MP5, MP6, MP7, MP8, MP9, MP10, MP11, MP12, MP13, MP14, MP15.

[0051] Example 2: Induced expression of engineered bacteria

[0052] The single colony on the plate was placed in LB liquid medium containing ampicillin antibiotics, and cultured at 37°C, 220rpm for 10 hours. This was the primary seed solution, which was inoculated into a new LB medium containing ampicillin antibiotics at a 1% inoculation amount and cultured at 37°C overnight. It was inoculated into a new LB medium containing ampicillin antibiotics at a 5% inoculation amount and cultured at 37°C for 2 hours. IPTG was added at a final concentration of 0.5mM and 18°C ​​for induction expression, and cultured for 16 hours. The bacteria were collected by centrifugation at 4000g and 4°C for 20 minutes.

[0053] After the cells were resuspended in 1X PBS and washed 2-3 times, an equal volume of lysis buffer (20mM Tris-HCl, 1mM EDTA, 500mM NaCl, pH8.5) was added for resuspending, 100X protease inhibitor PMSF was added, and the cells were dispersed using a high shear dispersing emulsifier. A high-pressure homogenizer was used at 800-1000 bar for homogenization for 15-30 min. After the cells were broken, 1mL of the broken liquid was taken and centrifuged at 4°C and 12000rpm for 2min. 80μL of the supernatant was taken, and the precipitate was resuspended with 200μL of lysis buffer, 120μL of the resuspended liquid was discarded, and 20μL of 5X protein loading buffer was added to both the supernatant and the precipitate. After mixing, the mixture was heated in a boiling water bath for 10min for SDS-PAGE to confirm protein expression.

[0054] Example 3: Purification of expression products

[0055] Purification of pET30a expression system: The bacteria were resuspended in lysis buffer, 100X protease inhibitor PMSF was added, and the bacteria were dispersed using a high-shear dispersing emulsifier. A high-pressure homogenizer was used at 800-1000 bar for homogenization for 15-30 min. After the cells were broken, the supernatant of the broken liquid obtained in Example 2 was heated in a 70°C water bath for 1 h to remove most of the impurities. After the protein liquid was heated, the temperature was lowered to room temperature, and the mixture was centrifuged at 4°C and 12000 rpm for 20 min. The supernatant was taken and the precipitate was discarded. 80 μL of the supernatant was added with 20 μL of 5X protein loading buffer, mixed, and heated in a boiling water bath for 10 min for SDS-PAGE, and then stained with Coomassie Brilliant Blue R-250. Imagej software was used to make a grayscale analysis gel map. The SDS-PAGE results are shown in FIG. Figure 4 The protein yield is shown in Table 5:

[0056] Table 5 Protein yield of pET30a expression system

[0057]

[0058]

[0059] Purification of MtuΔI-CM expression system: The cell pellets harvested in Example 2 that were confirmed to have protein expression were washed 2-3 times in 1XPBS, resuspended in buffer 1 (20mM Tris-HCl, 500mM NaCl, 1mM EDTA, pH8.5), and sonicated on ice using an ultrasonic crusher. The sample was centrifuged at 15,000g, 4°C for 20min, the precipitate was washed twice with buffer 1, and then resuspended in the same volume of buffer 2 (PBS buffer with 40mM Bis-Tris, 2mM EDTA, pH 6.2). Incubate at 25°C for 24h for internal mediated cleavage reaction. Then centrifuge to collect the soluble components, take 80μL of the supernatant and add 20μL of 5X protein loading buffer, mix and heat in a boiling water bath for 10min for SDS-PAGE, then stain with Coomassie Brilliant Blue R-250, and use imagej software to make grayscale analysis gel images. The SDS-PAGE results are as follows: Figure 4 As shown, the protein yield and recovery rate are shown in Table 6:

[0060] Table 6 Protein yield of MtuΔI-CM expression system

[0061]

[0062]

[0063]

[0064] It can be seen from Tables 5 and 6 that the yield of the target protein containing the functional peptide sequence in the functional peptide library is significantly higher than that of the target protein without functional peptide, and the recovery rate of the target protein containing the functional peptide sequence in the MtuΔI-CM expression system is significantly higher than that of the target protein containing the functional peptide (the recovery rate of the target protein refers to the percentage of the target protein obtained after in vitro cleavage of MtuΔI-CM to the fusion protein (L6KD-PT linker-MtuΔI-CM-functional peptide sequence-target protein) expressed by the entire system). Taking the target protein (COL-III) containing the functional peptide sequence SEQ ID N0.12 in the functional peptide library as an example, the supernatant protein solution of the target protein (COL-III) containing the functional peptide sequence SEQ ID N0.12 in the functional peptide library was taken for positive ion exchange resin enrichment purification (pET30a expression system) or membrane enrichment purification (MtuΔI-CM expression system), and the purified high-purity target protein COL-III containing the functional peptide sequence SEQ ID N0.12 was collected, 80 μL of supernatant was added to 20 μL of 5X protein loading buffer, and after mixing, it was heated in a boiling water bath for 10 min for SDS-PAGE, and the grayscale analysis gel map was made by imagej software. The protein yield and purity are shown in Table 7:

[0065] Table 7 Protein expression yield containing functional peptide sequence SEQ ID No. 12

[0066] protein Yield mg / L purity% L0-C(MtuΔI-CM) 584 97 L12-C(MtuΔI-CM) 754 97 L0-C (pET30a) 548 99 L12-C (pET30a) 651 99

[0067] The purification results of the target protein COL-III containing the functional peptide sequence SEQ ID NO.12 are as follows Figure 5 As shown, the target protein COL-III containing the functional peptide sequence SEQ ID N0.12 after purification by the pET30a expression system has a single band and high purity.

[0068] Example 4: Protein stability test

[0069] Taking III collagen (COL-III) as an example, the purified target protein solution containing functional peptides in the functional peptide library was concentrated to 500 mg / L, and the target protein without functional peptides at the same concentration was used as the control group. After being placed at room temperature for 60 days, 80 μL of the treated protein solution was added with 20 μL of 5X protein loading buffer, mixed and heated in a boiling water bath for 10 minutes for SDS-PAGE, and the grayscale analysis gel map was made using imagej software. The protein degradation rate is shown in Table 8:

[0070] Table 8 Protein degradation rate

[0071]

[0072]

[0073] As can be seen from the above table, the degradation rate of the target protein (COL-III) containing the functional peptides in the functional peptide library is significantly lower than that of the control, that is, the functional peptides in the functional peptide library of the present invention can significantly improve the stability of the protein. The results after the target protein solution containing the functional peptides was placed at room temperature for 60 days are as follows: Figure 6 As shown, the protein band is single, and the band of the control group has a tailing phenomenon (ie, the protein is degraded).

[0074] Example 5: Protein biological activity test

[0075] The purified fusion protein (FP) solution of the functional peptide in the library containing the functional peptide was concentrated to 500 mg / L, and the target protein without the functional peptide at the same concentration was used as the control group. 100 μL was taken and placed in a 1.5 mL centrifuge tube, and the luminescence was observed under ultraviolet light; 100 μL was taken and placed in a 96-well plate, and the fluorescence intensity was detected by a multifunctional microplate reader. The fluorescence intensity of the fusion protein was shown in FIG. Figure 7 and Table 9:

[0076] Table 9 Fluorescence intensity of fusion protein

[0077] protein Fluorescence intensity L0-FP (pET30a, control) 1135.33 L3-FP(MtuΔI-CM) 1124.27 L10-FP(MtuΔI-CM) 1131.41 L12-FP(MtuΔI-CM) 1112.13

[0078] Depend on Figure 7 As shown in Table 9, the biological activity of the target protein containing the functional peptides in the functional peptide library is basically consistent with that of the original protein, that is, the functional peptides in the functional peptide library of the present invention have basically no effect on the biological activity of the target protein.

Claims

1. A functional peptide, characterized in that Its sequence is shown in any one of SEQ ID No.1-15.

2. A gene encoding the functional peptide according to claim 1.

3. The coding gene according to claim 2, characterized in that Its base sequence is shown in any one of SEQ ID No.19-33.

4. Use of the functional peptide according to claim 1 in assisting the expression of a target protein.

5. An expression vector, characterized in that The vector comprises an expression gene for a recombinant protein, and the recombinant protein is obtained by adding the functional peptide according to claim 1 in front of the target protein.

6. A method for increasing protein production, characterized in that: The functional peptide according to claim 1 is fused with the target protein for expression.

7. Use of the functional peptide according to claim 1 in assisting the MtuΔI-CM protein expression system in expressing a target protein.

8. MtuΔI-CM expression vector, characterized in that The functional peptide according to claim 1 is inserted between the target protein and the intein.

9. A MtuΔI-CM expression host cell, which is obtained by transforming the MtuΔI-CM expression vector according to claim 8 into a host cell.

10. Use of the functional peptide according to claim 1 in improving protein stability.

Images