CN120098082B - A method and application for increasing protein yield - Google Patents

A method and application for increasing protein yield

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CN120098082B
CN120098082B CN202510321333.2A CN202510321333A CN120098082B CN 120098082 B CN120098082 B CN 120098082B CN 202510321333 A CN202510321333 A CN 202510321333A CN 120098082 B CN120098082 B CN 120098082B
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CN120098082A (en
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田江杰
旷哲伊
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Guangdong Puyan Biotechnology Co ltd
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Abstract

The invention discloses a functional peptide for improving the yield of recombinant proteins, the sequence of which is shown in any one of SEQ ID No. 1-15. Also discloses the coding gene and application thereof, and a corresponding method for improving the protein yield. The functional peptide can obviously improve the yield of recombinant proteins, can obviously reduce the loss rate of the recombinant proteins in the expression and purification processes of Mtu delta I-CM protein expression systems, reduces the production cost of the recombinant proteins, improves the stability of target proteins, is suitable for the production of various recombinant human proteins, lays a foundation for large-scale and low-cost mass production of various recombinant proteins, and has good commercial application prospect.

Description

Method for improving protein yield and application
Technical Field
The invention relates to a method for improving protein yield and application thereof, belonging to the technical field of protein expression.
Background
Recombinant proteins play a vital role in biomedical, cosmetic and food industries. The use of engineered microorganisms for the large-scale efficient production of recombinant proteins has attracted great interest due to the growing market demand. For this purpose, various expression systems have been developed, including prokaryotes, yeast, mammalian and human cells, plants and insects. Among them, E.coli is still the most widely used system due to its rapid growth, versatility and well-characterized genetic background. Although several recombinant protein bio-production processes have been commercially successful, challenges such as high cost and multi-step column purification required in downstream processing are faced. Therefore, how to realize efficient and large-scale expression of recombinant proteins, how to reduce separation cost and improve recovery efficiency is an important research topic of industrial biotechnology (J.Kaur et al International Journal of Biological Macromolecules,2018, 106:803-822).
Although protein purification techniques are now mature and widely used, many challenges remain, such as low recovery of recombinant proteins, poor stability, etc. Generally, 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 chromatographic columns such as affinity protein A chromatography, cation exchange chromatography, anion exchange chromatography and the like, and virus removal and inactivation are performed. After purification, the recombinant protein is replaced by ultrafiltration or diafiltration into a suitable buffer, and the recombinant protein is preserved by stock solution or by adding the final formulation ingredients to preserve the recombinant protein in the form of a semi-finished product. As these purification steps increase, the recovery of recombinant protein gradually decreases. In addition, during the whole production process, proteins undergo various damaging factors such as low pH, high salt, freeze thawing, light irradiation, shaking, shearing, and various (hydrophobic) surfaces, which may cause structural changes or degradation of the protein, affecting the stability of the protein.
Currently, column-free protein purification methods are considered to be the most recent effective alternative to conventional chromatographic techniques. The column-free purification method, i.e. the use of a polymeric tag to promote selective polymerization of the target protein during or after expression in the host cell, is recovered from the crude extract by simple centrifugation. Currently, a typical polymeric tag consists mainly of two parts, firstly, a sequence that is prone to aggregation and secondly, a cleavable site. The cleavable site is the removal of the aggregation-prone sequence by chemical means, protease-mediated means or intein-mediated cleavage. Recently, a cleavable self-aggregation tag scheme was developed for column-free purification of recombinant proteins, achieving higher yields and purities of recombinant proteins of interest. The self-aggregation tag regimen includes a self-assembling peptide, a PT linker, a pH-induced intron Mtu ΔI-CM, and a target protein. The fusion protein is expressed in the host cell in the form of insoluble aggregates, and the target protein is released into the supernatant in vitro by a pH change, thereby purifying the target protein. Currently, this self-aggregation tag scheme has been successfully applied to purification of recombinant proteins and peptides, such as human growth hormone, interferon alpha 2a and brain natriuretic peptide. However, this approach faces challenges, mainly involving premature cleavage of the fusion protein in the cell, resulting in a substantial decrease in yield (Z.Lin et al, aiche Journal,2020,66 (3): e 16806).
Studies have shown that although site-directed mutagenesis of Mtu ΔI-CM (H73Y/T430V, H V/T430S or H73V/T430C) has been demonstrated to reduce the premature cleavage rate of fusion proteins from 87% to 27-45% when lipase A production is performed using this protocol, the cleavage activity of Mtu ΔI-CM is regulated by the carboxy-terminal flanking residues (C+1 or C+2). When this scheme is used for the production of other proteins, the fusion protein still undergoes severe premature cleavage, and when the N-terminal residue of the protein of interest is cysteine (Cys), serine (Ser) or histidine (His), the rate of premature cleavage of the fusion protein in this scheme is as high as 79%. When different recombinant proteins are produced by using the system, different modification needs to be carried out on each recombinant protein, which not only increases the operation complexity but also increases the production cost.
Therefore, there remains a need in the art to develop more efficient recombinant protein expression purification strategies for use in low cost, simple, efficient recombinant protein production.
Disclosure of Invention
The invention aims to provide a functional peptide for improving the yield of recombinant proteins.
The invention adopts the technical scheme that:
The functional peptide has the sequence shown in any one of SEQ ID No.1-15, wherein the functional peptides shown in SEQ ID No. 9-15 are formed by combining the functional peptides shown in SEQ ID No. 0.1-8.
The base sequence of the coding gene of the functional peptide is shown in any one of SEQ ID No. 19-33.
The application of the functional peptide in assisting the expression of target proteins.
The expression vector comprises an expression gene of a recombinant protein, wherein the recombinant protein is formed by adding the functional peptide before a target protein, and the N end of the target protein is connected with the C end of the functional peptide.
The expression host cell is formed by transforming the expression vector into the host cell and is used for expressing recombinant proteins composed of functional peptides and target proteins.
A method for improving the yield of protein, which is to fuse and express the functional peptide and the target protein.
The application of the functional peptide in assisting Mtu delta I-CM protein expression system in expressing target protein. The Mtu delta I-CM protein expression system comprises self-aggregation peptide L6KD, PT type joint and pH induced self-assembly peptide Mtu delta I-CM, wherein the N end of the recombinant protein is connected with the C end of the pH induced self-assembly peptide Mtu delta I-CM through the functional peptide in the functional peptide library to form fusion protein.
Mtu DeltaI-CM expression vector, the functional peptide is inserted between the target protein and the intein.
Mtu DeltaI-CM expression host cells are obtained by transforming the Mtu DeltaI-CM expression vector into host cells and are used for expressing recombinant proteins consisting of functional peptides and target proteins.
The host cell is selected from the group consisting of prokaryotes, yeasts and higher eukaryotic cells, wherein the prokaryotes include bacteria of the genera Escherichia, bacillus, salmonella, pseudomonas and Streptomyces, preferably E.coli.
The application of the functional peptide in improving the protein stability.
The invention has the beneficial effects that:
The functional peptide disclosed by the invention can play a role in two different expression systems, can obviously improve the yield of recombinant proteins, can also obviously reduce the loss rate of the recombinant proteins in the Mtu delta I-CM protein expression system in the expression and purification processes, reduces the production cost of the recombinant proteins, improves the stability of target proteins, is suitable for the production of various recombinant humanized proteins, lays a foundation for large-scale and low-cost mass production of various recombinant proteins, and has good commercial application prospects.
Drawings
FIG. 1 shows a schematic structure of a protein expression system containing a functional peptide library and an expression vector map (pET 30a expression system).
FIG. 2 shows a schematic structural diagram of a protein expression system containing a functional peptide library and an expression vector map (Mtu ΔI-CM expression system).
FIG. 3 agarose gel electrophoresis of the construction of a protein expression system containing a functional peptide library. M: DL5000marker, the upper panel shows pET30a expression system 1:L0-C (control group ),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; without linker and the lower panel shows Mtu. DELTA.I-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.
FIG. 4 is a SDS-PAGE map of recombinant proteins based on expression of a protein expression system containing a functional peptide library. The upper panel shows pET30a expression system 1:L0-C (control group ),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; without linker) and the lower panel shows Mtu. DELTA.I-CM expression system 1:M-L0-C (control group ),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：III type collagen (COL-III) without linker), B Fibronectin (FN), C Fusion Protein (FP).
FIG. 5 SDS-PAGE of functional peptide-containing recombinant protein COL-III after enrichment purification. M protein Marker,1:L12-C (Mtu ΔI-CM expression system), 2:L0-C (Mtu ΔI-CM expression system), 3:L0-C (pET 30a expression system), 4:L12-C (pET 30a expression system), 5:0.5mg/ml bovine serum albumin.
FIG. 6 is a SDS-PAGE graph of recombinant protein stability test after purification based on a protein expression system containing a functional peptide library. M is a protein Marker,1 is a COL-III sample containing a functional peptide L3 after treatment (60 days at room temperature, pET30a expression system), 2 is a COL-III sample containing a functional peptide L10 after treatment (60 days at room temperature, pET30a expression system), 3 is a COL-III sample containing a functional peptide L12 after treatment (60 days at room temperature, pET30a expression system), 4 is a COL-III sample containing a functional peptide L15 after treatment (60 days at room temperature, pET30a expression system), 5 is a COL-III sample containing no functional peptide after treatment (60 days at room temperature, control), 6 is a COL-III sample containing a functional peptide L3 after treatment (60 days at room temperature, mtu. DELTA.I-CM expression system), 8 is a COL-III sample containing a functional peptide L10 after treatment (60 days at room temperature, 35. DELTA.I-35 days at room temperature, DELTA.I-III expression system), and 35 is a COL-III sample containing a functional peptide L10 after treatment (35 days at room temperature, DELTA.I-III expression system).
FIG. 7 shows green fluorescence of Fusion Proteins (FPs) containing functional peptides under UV irradiation. 1:L0-FP (pET 30a, control), 2:L3-FP (Mtu ΔI-CM), 3:L10-FP (Mtu ΔI-CM), 4:L12-FP (Mtu ΔI-CM).
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting. Specific materials and sources thereof used in embodiments of the present invention are provided below. It will be understood that these are merely exemplary and are not intended to limit the invention, as materials identical or similar to the type, model, quality, nature or function of the reagents and instruments described below may be used in the practice of the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 preparation of plasmid for protein expression System containing functional peptide library
(1) Gene design and synthesis
In the invention, a recombinant protein expression vector containing functional peptides L1-L15 in a functional peptide library is constructed, human type III collagen (COL-III), fibronectin (FN) and Fusion Protein (FP) formed by connecting fibronectin and green fluorescent protein through PTlinker are selected as target proteins for testing, the corresponding amino acid sequences are shown as SEQ ID No.16-18, the amino acid sequences of the functional peptides L1-L15 in the functional peptide library are shown as SEQ ID No. 1-SEQ ID No.15, and the corresponding coding gene sequences are shown as SEQ ID No. 19-SEQ ID No. 33. The functional peptide L1-L15, the corresponding coding nucleotide sequence of the recombinant protein COL-III, FN, FP and the Gibson assembly primer are synthesized by the process of the engineering.
(2) Construction of recombinant expression vectors
And (3) carrying out PCR amplification by taking the synthesized gene fragment as a template, wherein a ① pET30a expression system is obtained by connecting a target gene fragment containing the functional peptide in the functional peptide library and a pET30a expression vector skeleton through Gibson assembly, so as to obtain corresponding plasmids, wherein the primer sequences are shown in a table 1, and the plasmid map is shown in a figure 1. ② Mtu delta I-CM expression system, namely connecting a Mtu delta I-CM expression frame, a target gene fragment containing functional peptide in a functional peptide library and a pET30a expression vector skeleton through Gibson assembly to obtain corresponding plasmids, wherein primer sequences are shown in table 2, plasmid maps are shown in figure 2, and L0 refers to a control, namely target protein without the functional peptide.
TABLE 1 primer sequences for pET30a expression systems
TABLE 2 primer sequences for Mtu DeltaI-CM expression systems
TABLE 3PCR amplification System
After the PCR system shown in Table 3 was prepared, the mixture was homogenized and centrifuged, and the PCR amplification conditions were a first stage of 98℃pre-denaturation for 30s, a second stage of 98℃denaturation for 10s, a 50-72℃annealing for 30s, a 72℃extension for 30s/kb,32 cycles, and a third stage of 72℃final extension for 2min. The above vectors and gene fragments were recovered using a universal DNA purification kit (Tiangen Biochemical Co., ltd.) and carried out according to the procedure of the product instruction.
TABLE 4Gibson ligation System
System components Volume of the composition
Gibson Assembly Master Mix(2X) 5μL
Ligation fragment 0.2–1pmols*XμL
dd H2O (5-X)μL
Total system 10μL
The above ingredients were mixed on ice and then placed in a 37 ℃ hot bath for 60min, and the resulting ligation product was stored on ice or at-20 ℃ for subsequent competent transformations.
The ligation product was transformed into host E.coli DH5α by heat shock method, plated on LB culture resistant plates, cultured overnight in a 37℃incubator, and the monoclonal was picked for colony PCR, and the results are shown in FIG. 3. Transferring the preliminarily obtained positive clone to LB liquid medium, culturing overnight at 37 ℃ in a shaking table and 220rpm, extracting plasmids by a plasmid rapid extraction kit, and respectively naming the successfully constructed plasmids as pET30a expression systems, namely L0-C, L1-C (functional peptide L1-COL-III), L2-C, L-C, L4-C, L5-C, L6-C, L7-C, L-C, L9-C, L-C, L11-C, L12-C, L13-C, L14-C, L15-C; L0-C, L-1-F (functional peptide L1-FN), 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-F, L0-C, L1-P (functional peptide L1-FP), 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-P, 3932ΔI-CM expression System M-L0-C, L-L1-C (3932ΔI-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-L6-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.
(3) Construction of engineering bacteria
Transferring the recombinant expression plasmid into competent cells of Escherichia coli BL21 (DE 3) by chemical transformation, and performing antibiotic plate screening to obtain positive engineering bacteria, wherein ① comprises collecting 4 μl recombinant expression plasmid into 100 μl of competent cells BL21 (DE 3), standing on ice for 30min, ② heating the mixture in a water bath at 42deg.C for 90s, rapidly standing on ice for 2min, ③ adding 900 μl of non-resistant LB liquid medium (10 g/L peptone, 5g/L yeast extract, 10g/L sodium chloride) into the mixture, culturing at 37deg.C for 1 hr at constant temperature, ④ comprises uniformly coating 200 μl of the bacterial liquid onto LB solid medium plate (10 g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100 μg/mL ampicillin) containing ampicillin, culturing at constant temperature in a shaking incubator at temperature of ⑤, culturing at about 16 ° for 53 a clear colony expression system of the strain in the shaking table system of ：C0、C1、C2、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 A, and obtaining clear expression system of the strain ：M0、M1、M2、M3、M4、M5、M6、M7、M8、M9、M10、M11、M12、M13、M14、M15;MF0、MF1、MF2、MF3、MF4、MF5、MF6、MF7、MF8、MF9、MF10、MF11、MF12、MF13、MF14、MF15;MP0、MP1、MP2、MP3、MP4、MP5、MP6、MP7、MP8、MP9、MP10、MP11、MP12、MP13、MP14、MP15.
Example 2 Induction expression of engineering bacteria
The single colony on the plate was placed in LB liquid medium containing ampicillin at 37℃and 220rpm for 10 hours, which was first seed liquid, inoculated in a new LB medium containing ampicillin at 1% of the inoculum size, cultured overnight at 37℃and in a new LB medium containing ampicillin at 5% of the inoculum size, cultured for 2 hours at 37℃and induced to express by adding IPTG at a final concentration of 0.5mM and at 18℃for 16 hours. The cells were collected by centrifugation at 4000g and 4℃for 20 min.
After re-suspending the cells in 1 XPBS for 2-3 times, adding an equal volume of lysis buffer (20 mM Tris-HCl,1mM EDTA,500mM NaCl,pH8.5) for re-suspension, adding a 100 Xprotease inhibitor PMSF, dispersing the cells by using a high-shear dispersion emulsifier, homogenizing for 15-30min by using a high-pressure homogenizer at 800-1000bar, crushing the cells, taking 1mL of crushed solution, centrifuging for 2min at 4 ℃ and 12000rpm, taking 80 mu L of supernatant, re-suspending the sediment by 200 mu L of lysis buffer, discarding 120 mu L of re-suspension, adding 20 mu L of 5 Xprotein loading buffer into the supernatant and the sediment, heating for 10min in a boiling water bath after mixing, and performing SDS-PAGE to confirm protein expression.
EXAMPLE 3 purification of expression products
Purifying a pET30a expression system, namely suspending the bacterial body in a lysis buffer solution, adding a 100X protease inhibitor PMSF, dispersing the bacterial body by using a high shear dispersing emulsifier, homogenizing for 15-30min by using a high pressure homogenizer at 800-1000bar, crushing cells, heating the supernatant of the crushed liquid obtained in the example 2 in a 70 ℃ water bath for 1h to remove most of the impurity proteins, cooling the heated protein liquid to room temperature, centrifuging at 4 ℃ and 12000rpm for 20min, taking the supernatant, discarding the precipitate, wherein 80 mu L of the supernatant is added into 20 mu L of a 5X protein loading buffer solution, heating for 10min in a boiling water bath for SDS-PAGE after mixing, then dyeing by using Coomassie brilliant blue R-250, and carrying out gray analysis gel diagram by imagej software, wherein the SDS-PAGE result is shown in figure 4, and the protein yield is shown in Table 5:
TABLE 5 protein yield of pET30a expression System
Mtu. DELTA.I-CM expression System purification the cell pellet determined to have protein expression obtained in example 2 was washed 2-3 times with 1XPBS, resuspended in buffer 1 (20 mM Tris-HCl,500mM NaCl,1mM EDTA,pH 8.5) and sonicated on ice using a sonicator. The sample was centrifuged at 15,000g at 4℃for 20min, and the pellet was washed twice with buffer 1 and then resuspended in the same volume of buffer 2 (40 mM Bis-Tris,2mM EDTA, pH 6.2 added to PBS buffer). Incubation was performed at 25 ℃ for 24h, with an internally mediated cleavage reaction. Then, the soluble components were collected by centrifugation, 80. Mu.L of the supernatant was added to 20. Mu.L of 5 Xprotein loading buffer, mixed and heated in a boiling water bath for 10min for SDS-PAGE, and then stained with Coomassie Brilliant blue R-250, and imagej software was used for gray scale analysis gel, the SDS-PAGE results are shown in FIG. 4, and the protein yields and recovery rates are shown in Table 6:
TABLE 6Mtu protein yield of DeltaI-CM expression System
As can be seen from tables 5 and 6, the yield of the target protein containing the functional peptide sequence in the functional peptide library was significantly higher than that of the target protein without the functional peptide, and the recovery rate of the target protein containing the functional peptide sequence in the Mtu ΔI-CM expression system was significantly higher than that of the target protein containing the functional peptide (the target protein recovery rate means the percentage of the target protein obtained after Mtu ΔI-CM cleavage in vitro, which is expressed as a fusion protein (L6 KD-PT linker-Mtu ΔI-CM functional peptide sequence-target protein) of the whole system). Taking a target protein (COL-III) containing a functional peptide sequence SEQ ID N0.12 in a functional peptide library as an example, taking a target protein (COL-III) supernatant containing the functional peptide sequence SEQ ID N0.12 in the functional peptide library for positive ion exchange resin enrichment purification (pET 30a expression system) or filter membrane enrichment purification (Mtu DeltaI-CM expression system), collecting purified target protein COL-III with high purity and functional peptide sequence SEQ ID N0.12, taking 80 mu L of supernatant, adding 20 mu L of 5X protein loading buffer, heating in a boiling water bath for 10min after mixing, performing SDS-PAGE, and imagej software as a gray scale analysis gel diagram, wherein the protein yield and purity are shown in Table 7:
TABLE 7 protein expression yield with functional peptide sequence SEQ ID N0.12
Proteins 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
The purification result of the target protein COL-III containing the functional peptide sequence SEQ ID N0.12 is shown in figure 5, and the target protein COL-III containing the functional peptide sequence SEQ ID N0.12 after the purification of the pET30a expression system has single band and high purity.
Example 4 protein stability test
Taking collagen III (COL-III) as an example, concentrating the purified target protein solution containing the functional peptide in the functional peptide library to 500mg/L, taking the target protein without the functional peptide with the same concentration as a control group, standing at normal temperature for 60 days, taking 80 mu L of the treated protein solution, adding 20 mu L of 5X protein loading buffer solution, mixing, heating in a boiling water bath for 10min for SDS-PAGE, and carrying out a gray analysis gel diagram by imagej software, wherein the protein degradation rate is shown in Table 8:
TABLE 8 protein degradation Rate
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, i.e., the functional peptides in the functional peptide library of the invention can significantly improve the stability of the protein. The result of the functional peptide-containing target protein solution after being left at normal temperature for 60 days is shown in FIG. 6, the protein bands are single, wherein the bands of the control group are trailing (i.e. the protein is degraded).
Example 5 protein bioactivity assay
Concentrating the purified Fusion Protein (FP) solution containing the functional peptide in the library to 500mg/L, taking the target protein without the functional peptide with the same concentration as a control group, respectively taking 100 mu L of the target protein into a 1.5mL centrifuge tube, and placing the target protein into ultraviolet light for irradiation and observation of the luminescence condition, respectively taking 100 mu L of the target protein into a 96-well plate, and detecting the fluorescence intensity of the target protein by a multifunctional enzyme-labeled instrument, wherein the fluorescence intensity of the fusion protein is shown in FIG. 7 and Table 9:
TABLE 9 fluorescence intensity of fusion proteins
Proteins Fluorescence intensity
L0-FP (pET 30a, control) 1135.33
L3-FP(MtuΔI-CM) 1124.27
L10-FP(MtuΔI-CM) 1131.41
L12-FP(MtuΔI-CM) 1112.13
As can be seen from fig. 7 and table 9, the biological activity of the target protein containing the functional peptides in the functional peptide library is substantially identical to that of the original protein, i.e., the functional peptides in the functional peptide library of the present invention have substantially no influence on the biological activity of the target protein.

Claims (10)

1.一种功能肽，其特征在于，其序列如SEQ ID No.12所示。1. A functional peptide, characterized in that its sequence is shown in SEQ ID No. 12. 2.权利要求1所述的功能肽的编码基因。2. The gene encoding the functional peptide of claim 1. 3.根据权利要求2所述的编码基因，其特征在于，其碱基序列如SEQ ID No.30所示。3. The encoding gene according to claim 2, characterized in that its base sequence is as shown in SEQ ID No. 30. 4.权利要求1所述的功能肽在辅助目的蛋白表达中的应用。4. The use of the functional peptide according to claim 1 in assisting the expression of the target protein. 5.表达载体，其特征在于，所述载体包括重组蛋白的表达基因，所述重组蛋白为在目的蛋白前加上权利要求1所述的功能肽而成。5. An expression vector, characterized in that the vector comprises an expression gene for a recombinant protein, wherein the recombinant protein is formed by adding the functional peptide of claim 1 before the target protein. 6.一种提高蛋白产量的方法，其特征在于，将权利要求1所述的功能肽与目的蛋白融合表达。6. A method for increasing protein yield, characterized in that the functional peptide of claim 1 is fused with the target protein for expression. 7.权利要求1所述的功能肽在辅助MtuΔI-CM蛋白表达体系表达目的蛋白中的应用。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.MtuΔI-CM表达载体，其特征在于，在目的蛋白与内含肽之间插入权利要求1所述的功能肽。8. The MtuΔI-CM expression vector, characterized in that the functional peptide of claim 1 is inserted between the target protein and the integrin. 9.MtuΔI-CM表达宿主细胞，为将权利要求8所述的MtuΔI-CM表达载体转化到宿主细胞中而成。9. MtuΔI-CM expression host cells are prepared by transforming the MtuΔI-CM expression vector of claim 8 into host cells. 10.权利要求1所述的功能肽在提高蛋白稳定性中的应用。10. The use of the functional peptide of claim 1 in improving protein stability.
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