A method and application for increasing protein yield

Abstract

This invention discloses a functional peptide for increasing recombinant protein yield, the sequence of which is shown in any of SEQ ID No. 1-15. The encoding gene and its applications are also disclosed, along with corresponding methods for increasing protein yield. The functional peptide of this invention can significantly increase the yield of recombinant proteins, significantly reduce the loss rate of recombinant proteins during expression and purification in the MtuΔI-CM protein expression system, reduce the production cost of recombinant proteins, and improve the stability of the target protein. It is suitable for the production of various recombinant human proteins, laying the foundation for large-scale, low-cost mass production of various recombinant proteins and possessing good commercial application prospects.

Description

Technical Field

[0001] This invention relates to a method and application for increasing protein yield, belonging to the field of protein expression technology. Background Technology

[0002] Recombinant proteins play a crucial role in the biomedical, cosmetic, and food industries. Due to increasing market demand, the large-scale, efficient production of recombinant proteins using engineered microorganisms has attracted considerable interest. Various expression systems have been developed for this purpose, including those from prokaryotes, yeast, mammalian and human cells, plants, and insects. Among these, *E. coli* remains the most widely used system due to its rapid growth, versatility, and well-defined genetic background. Although several recombinant protein bioproduction processes have achieved commercial success, challenges remain, such as high costs and the need for multi-step column purification in downstream processing. Therefore, achieving efficient and large-scale expression of recombinant proteins, and reducing separation costs while improving 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, many challenges remain, such as low recombinant protein recovery rates and poor stability. Typically, after recombinant proteins are synthesized by microorganisms, insoluble cell residues are removed primarily through centrifugation or filtration. The supernatant containing the recombinant protein is then purified using several chromatographic steps, including affinity protein A chromatography, cation exchange chromatography, and anion exchange chromatography, while also removing and inactivating viruses. After purification, the recombinant protein is replaced with an appropriate buffer solution using ultrafiltration or percolation, and then stored as a stock solution or added to the final formulation components for preservation as a semi-finished product. With each additional purification step, the recombinant protein recovery rate gradually decreases. Furthermore, throughout the production process, proteins are subjected to various destructive factors, such as low pH, high salt, freeze-thaw cycles, light exposure, vibration, shearing, and various (hydrophobic) surfaces. These factors can all cause structural changes or degradation of the protein, affecting its stability.

[0004] Currently, column-free protein purification methods are considered an effective alternative to traditional chromatography techniques. Column-free purification utilizes polymeric tags to promote the selective polymerization of target proteins during or after expression in host cells, followed by recovery from the crude extract via simple centrifugation. Typical polymeric tags currently consist of two parts: first, an easily aggregated sequence, and second, a cleavable site. The cleavable site is the easily aggregated sequence removed by chemical methods, protease-mediated methods, or intron-mediated cleavage. Recently, a cleavable self-aggregating tag scheme has been developed for column-free purification of recombinant proteins, achieving high yields and purity of the target recombinant protein. This self-aggregating tag scheme includes a self-assembling peptide, a PT linker, a pH-induced intron MtuΔI-CM, and the target protein. The fusion protein is expressed in host cells as insoluble aggregates, and the target protein is released in vitro into the supernatant via pH changes, thereby purifying the target protein. This self-aggregating 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 intracellular cleavage of the fusion protein, leading to a significant decrease in yield (Z. Lin et al., Aiche Journal, 2020, 66(3):e16806).

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

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

[0007] The purpose of this invention is to provide a functional peptide that can improve the yield of recombinant proteins.

[0008] The technical solution adopted in this invention is as follows:

[0009] A functional peptide for increasing the yield of recombinant proteins, the sequence of which is shown in any one of SEQ ID No. 1-15, wherein the functional peptides shown in SEQ ID No. 9 to SEQ ID No. 15 are formed by combining two functional peptides shown in SEQ ID No. 1 to SEQ ID No. 8.

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

[0011] The aforementioned functional peptides are used to assist in the expression of target proteins.

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

[0013] The expression host cell is formed by converting 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 to increase protein yield involves fusing the aforementioned functional peptide with the target protein for expression.

[0015] The aforementioned functional peptides are used in the expression of the target protein in the MtuΔI-CM protein expression system. The MtuΔI-CM protein expression system comprises the self-aggregating peptide L6KD, a PT-type linker, and a pH-inducible self-assembling peptide MtuΔI-CM; the N-terminus of the recombinant protein is linked to the C-terminus of the pH-inducible self-assembling peptide MtuΔI-CM via a functional peptide from the aforementioned functional peptide library to form a fusion protein.

[0016] The MtuΔI-CM expression vector inserts the aforementioned functional peptide between the target protein and its integrins.

[0017] MtuΔI-CM expression host cells are formed by transforming the aforementioned MtuΔI-CM expression vector into host cells, and are used to express recombinant proteins composed of functional peptides and target proteins.

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

[0019] The aforementioned functional peptides are used to improve protein stability.

[0020] The beneficial effects of this invention are:

[0021] The functional peptides of this invention can function in two different expression systems, significantly increasing the yield of recombinant proteins and significantly reducing the loss rate of recombinant proteins during expression and purification in the MtuΔI-CM protein expression system, thus reducing the production cost of recombinant proteins and improving the stability of the target protein. They are suitable for the production of various recombinant human proteins, laying the foundation for large-scale, low-cost mass production of various recombinant proteins and showing good commercial application prospects. Attached Figure Description

[0022] Figure 1 A schematic diagram of the structure of the protein expression system containing the functional peptide library and an image of the expression vector (pET30a expression system).

[0023] Figure 2 A schematic diagram of the structure of a protein expression system containing a functional peptide library and an image of the expression vector (MtuΔI-CM expression system).

[0024] Figure 3 Agarose gel electrophoresis images of the protein expression system constructed with functional peptide libraries. M: DL5000 marker. The top image shows 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 bottom image shows 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.

[0025] Figure 4SDS-PAGE images of recombinant proteins expressed using protein expression systems containing functional peptide libraries. The top image shows 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 bottom image shows the MtuΔI-CM expression system: 1 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 image of 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 images of the stability of recombinant proteins after purification, based on protein expression systems containing functional peptide libraries. M: Protein Marker; 1: COL-III sample containing functional peptide L3 after treatment (stored at room temperature for 60 days, pET30a expression system); 2: COL-III sample containing functional peptide L10 after treatment (stored at room temperature for 60 days, pET30a expression system); 3: COL-III sample containing functional peptide L12 after treatment (stored at room temperature for 60 days, pET30a expression system); 4: COL-III sample containing functional peptide L15 after treatment (stored at room temperature for 60 days, pET30a expression system); 5: COL-III sample without functional peptides after treatment (stored at room temperature for 60 days, control). 6: COL-III sample without functional peptide after treatment (stored at room temperature for 60 days, control); 7: COL-III sample containing functional peptide L3 after treatment (stored at room temperature for 60 days, MtuΔI-CM expression system); 8: COL-III sample containing functional peptide L10 after treatment (stored at room temperature for 60 days, MtuΔI-CM expression system); 9: COL-III sample containing functional peptide L12 after treatment (stored at room temperature for 60 days, MtuΔI-CM expression system); 10: COL-III sample containing functional peptide L15 after treatment (stored at room temperature for 60 days, MtuΔI-CM expression system).

[0028] Figure 7 The functional peptide fusion protein (FP) fluoresces green under ultraviolet light. 1: L0-FP (pET30a, control), 2: L3-FP (MtuΔI-CM), 3: L10-FP (MtuΔI-CM), 4: L12-FP (MtuΔI-CM). Detailed Implementation

[0029] The present invention is further illustrated below by way of examples, but is not intended to limit the invention. 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 not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or function as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following examples are commercially available.

[0030] Example 1: Preparation of plasmids for protein expression systems containing functional peptide libraries

[0031] (1) Gene design and synthesis

[0032] In this invention, a recombinant protein expression vector containing functional peptides L1-L15 from a functional peptide library was constructed. Human type III collagen (COL-III), fibronectin (FN), and a fusion protein (FP) formed by linking fibronectin and green fluorescent protein via a PTlinker were selected as the target proteins for testing. Their corresponding amino acid sequences are shown in SEQ ID No. 16-18. The amino acid sequences of functional peptides L1-L15 from 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 corresponding coding nucleotide sequences of functional peptides L1-L15, recombinant proteins COL-III, FN, and FP, as well as Gibson assembly primers, were synthesized bioengineering.

[0033] (2) Construction of recombinant expression vectors

[0034] Using the synthesized gene fragment as a template, PCR amplification was performed. ① pET30a expression system: The target gene fragment containing the functional peptide from the functional peptide library and the pET30a expression vector backbone were ligated using Gibson assembly to obtain the corresponding plasmid. The primer sequences are shown in Table 1, and the plasmid map is shown in Table 2. Figure 1 As shown in Table 2. ②MtuΔI-CM Expression System: The MtuΔI-CM expression framework, the target gene fragment containing the functional peptide from the functional peptide library, and the pET30a expression vector backbone were ligated using Gibson assembly to obtain the corresponding plasmids. The primer sequences are shown in Table 2, and the plasmid map is shown in Table 3. Figure 2 As shown, L0 refers to the control, which is the target protein without the addition of functional peptides.

[0035] Table 1 Primer sequences for the pET30a expression system

[0036]

[0037]

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

[0039]

[0040]

[0041] Table 3 PCR amplification system

[0042]

[0043]

[0044] After preparing the PCR system as shown in Table 3, mix well and centrifuge. The PCR amplification conditions are as follows: Stage 1: 98℃ pre-denaturation for 30s; Stage 2: 98℃ denaturation for 10s, 50-72℃ annealing for 30s, 72℃ extension for 30s / kb, 32 cycles; Stage 3: 72℃ final extension for 2min. The vectors and gene fragments were recovered using a universal DNA purification kit (Tiangen Biotech Co., Ltd.), following the instructions in the product manual.

[0045] Table 4 Gibson Connection System

[0046] System components Component volume Gibson Assembly Master Mix(2X) 5μL Connecting fragments 0.2–1 pmols*XμL <![CDATA[dd H2O]]> (5-X)μL Overall system 10μL

[0047] After mixing the above components on ice, place them in a 37°C heat bath for 60 minutes. Store the resulting ligation product on ice or at -20°C for subsequent competent cell transformation.

[0048] The ligation product was transformed into the host bacterium *E. coli* DH5α using a heat shock method, plated onto LB agar plates, and incubated overnight at 37°C. Single colonies were picked for colony PCR, and the results are as follows: Figure 3As shown. The initially obtained positive clones were transferred to LB liquid medium and cultured overnight in a shaker at 37°C and 220 rpm. Plasmids were extracted using a rapid plasmid extraction kit, and the successfully constructed plasmids were named as follows: 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 The following peptide systems are used: 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 transformed into *E. coli* BL21(DE3) competent cells via chemical transformation. Positive engineered bacteria were obtained by screening with antibiotic plates. The specific procedure was as follows: ① 4 μL of the recombinant expression plasmid was added to 100 μL of *E. coli* BL21(DE3) competent cells and incubated on ice for 30 min; ② The mixture was heat-shocked in a 42℃ water bath for 90 s, then quickly placed on ice for 2 min; ③ 900 μL of antibiotic-free LB liquid medium (10 g / L peptone, 5 g...) was added to the mixture. ④ Incubate the bacterial culture at 37℃ and 220 rpm for 1 hour using a shaker (10 g / L yeast extract, 10 g / L sodium chloride). ⑤ Spread 200 μL of this bacterial culture evenly onto an LB agar plate containing ampicillin (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 100 μg / mL ampicillin). ⑥ Invert the plate and incubate at 37℃ for approximately 16 hours until clearly visible colonies appear. The corresponding expression system for the engineered strain pET30a is obtained: C0, C1, C2, C3, C4, C5, C6, C7, C8, C9 ... 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] Single colonies from the above plates were placed in LB broth containing ampicillin and incubated at 37°C and 220 rpm for 10 hours. This was the primary seed culture. A 1% inoculum was then added to fresh LB broth containing ampicillin and incubated overnight at 37°C. A 5% inoculum was then added to fresh LB broth containing ampicillin and incubated at 37°C for 2 hours. IPTG was then added to a final concentration of 0.5 mM and incubated at 18°C ​​for 16 hours to induce expression. The cells were then collected by centrifugation at 4000g and 4°C for 20 minutes.

[0053] After resuspending the bacterial cells in 1X PBS and washing 2-3 times, resuspend them in an equal volume of lysis buffer (20mM Tris-HCl, 1mM EDTA, 500mM NaCl, pH 8.5). Add 100X protease inhibitor PMSF and disperse the bacterial cells using a high-shear dispersing emulsifier. Homogenize using a high-pressure homogenizer at 800-1000 bar for 15-30 min. After cell lysis, take 1 mL of the lysate and centrifuge at 4℃ and 12000 rpm for 2 min. Take 80 μL of supernatant and resuspend the precipitate in 200 μL of lysis buffer. Discard 120 μL of the resuspended precipitate. Add 20 μL of 5X protein loading buffer to both the supernatant and the precipitate, mix, and heat in a boiling water bath for 10 min before performing SDS-PAGE to confirm protein expression.

[0054] Example 3: Purification of the expression product

[0055] Purification of pET30a expression system: Bacterial cells were resuspended in lysis buffer, and 100X protease inhibitor PMSF was added. The cells were dispersed using a high-shear dispersing emulsifier and homogenized at 800-1000 bar for 15-30 min using a high-pressure homogenizer. After cell lysis, the supernatant obtained in Example 2 was heated in a 70°C water bath for 1 h to remove most of the impurities. After heating, the protein solution was cooled to room temperature and centrifuged at 4°C and 12000 rpm for 20 min. The supernatant was collected, and the precipitate was discarded. 80 μL of the supernatant was added to 20 μL of 5X protein loading buffer, mixed, and heated in a boiling water bath for 10 min for SDS-PAGE. Staining was then performed using Coomassie Brilliant Blue R-250, and grayscale analysis gel images were generated using ImageJ software. The SDS-PAGE results are shown below. Figure 4 As shown in Table 5, protein yield is as follows:

[0056] Table 5 Protein yield of the pET30a expression system

[0057]

[0058]

[0059] Purification of the MtuΔI-CM expression system: Cell particles with confirmed protein expression obtained in Example 2 were washed 2-3 times with 1X PBS, resuspended in buffer 1 (20mM Tris-HCl, 500mM NaCl, 1mM EDTA, pH 8.5), and sonicated on ice using an ultrasonic homogenizer. The sample was centrifuged at 15,000g, 4°C for 20 min. 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). Incubation was performed at 25°C for 24 h to induce internally mediated lysis. The soluble fraction was then collected by centrifugation. 80 μL of the supernatant was added to 20 μL of 5X protein loading buffer, mixed, and heated in a boiling water bath for 10 min for SDS-PAGE. Staining was performed with Coomassie Brilliant Blue R-250, and grayscale analysis was performed using ImageJ software. The SDS-PAGE results are shown below. Figure 4 As shown in Table 6, protein yield and recovery rate are as follows:

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

[0061]

[0062]

[0063]

[0064] As shown in Tables 5 and 6, the yield of target proteins containing functional peptide sequences from the functional peptide library was significantly higher than that of target proteins without functional peptides. In the MtuΔI-CM expression system, the recovery rate of target proteins containing functional peptide sequences was significantly higher than that of target proteins containing functional peptides (target protein recovery rate refers to the percentage of target protein obtained after in vitro lysis of MtuΔI-CM out of the total fusion protein expressed in the system (L6KD-PT linker-MtuΔI-CM-functional peptide sequence-target protein)). Taking the target protein (COL-III) containing the functional peptide sequence SEQ ID NO.12 from the functional peptide library as an example, the supernatant protein solution of the target protein (COL-III) containing the functional peptide sequence SEQ ID NO.12 from the functional peptide library was enriched and purified by positive ion exchange resin (pET30a expression system) or by filter membrane enrichment and purification (MtuΔI-CM expression system). The purified target protein COL-III containing the functional peptide sequence SEQ ID NO.12 with high purity was collected. 80 μL of supernatant was added to 20 μL of 5X protein loading buffer, mixed, and heated in a boiling water bath for 10 min for SDS-PAGE. Grayscale analysis gel images were generated using imagej software. The protein yield and purity are shown in Table 7.

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

[0066] protein Production (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 NO.12, purified from the pET30a expression system, exhibits a single band and high purity.

[0068] Example 4: Protein stability test

[0069] Taking collagen III (COL-III) as an example, the purified target protein solution containing functional peptides from the functional peptide library was concentrated to 500 mg / L. A control group containing the same concentration of target protein without functional peptides was used. After 60 days at room temperature, 80 μL of the treated protein solution was added to 20 μL of 5X protein loading buffer. After mixing, the mixture was heated in a boiling water bath for 10 min for SDS-PAGE. Grayscale analysis of the gel image was performed using ImageJ software. The protein degradation rate is shown in Table 8.

[0070] Table 8 Protein degradation rate

[0071]

[0072]

[0073] As shown in the table above, the degradation rate of the target protein (COL-III) containing the functional peptides from the functional peptide library was significantly lower than that of the control, indicating that the functional peptides in the functional peptide library of this invention can significantly improve protein stability. The results of the target protein solution containing the functional peptides after being stored at room temperature for 60 days are as follows: Figure 6 As shown, the protein bands are single, while the bands in the control group show tailing (i.e., protein degradation).

[0074] Example 5: Protein Bioactivity Assay

[0075] The purified fusion protein (FP) solution containing functional peptides from the library was concentrated to 500 mg / L. A control group containing the same concentration of the target protein without functional peptides was used. 100 μL of each fusion protein was placed in a 1.5 mL centrifuge tube and irradiated under UV light to observe its luminescence. 100 μL of each fusion protein was also placed in a 96-well plate, and its fluorescence intensity was detected using a multi-mode microplate reader. The fluorescence intensity of the fusion protein is shown in the figure below. Figure 7 And Table 9:

[0076] Table 9 Fluorescence intensity of fusion proteins

[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 bioactivity of the target protein containing the functional peptides from the functional peptide library is basically the same as that of the original protein, meaning that the functional peptides in the functional peptide library of this invention have basically no effect on the bioactivity of the target protein.

Claims

1. A functional peptide, characterized in that, Its sequence is shown in SEQ ID No.

12.

2. The gene encoding the functional peptide of claim 1.

3. The encoding gene according to claim 2, characterized in that, Its base sequence is shown in SEQ ID No.

30.

4. The use of the functional peptide according to claim 1 in assisting the expression of the target protein.

5. An expression vector, characterized in that, The vector includes an expression gene for a recombinant protein, which is formed by adding the functional peptide of claim 1 before the target protein.

6. A method for increasing protein yield, characterized in that, The functional peptide described in claim 1 is fused with the target protein for expression.

7. The application of the functional peptide according to claim 1 in the expression of the target protein in the MtuΔI-CM protein expression system.

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

9. MtuΔI-CM expression host cells are prepared by transforming the MtuΔI-CM expression vector of claim 8 into host cells.

10. The use of the functional peptide of claim 1 in improving protein stability.

Images