CN114480460B

CN114480460B - Method for simultaneously enhancing expression quantity and solubility of target protein in prokaryotic system

Info

Publication number: CN114480460B
Application number: CN202210107465.1A
Authority: CN
Inventors: 高建华; 欧阳春平; 赵娟丽; 王兴春
Original assignee: Shanxi Agricultural University
Current assignee: Shanxi Agricultural University
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2023-06-16
Anticipated expiration: 2042-01-28
Also published as: CN114480460A

Abstract

The present invention discloses a method for simultaneously enhancing the expression amount and solubility of a target protein in a prokaryotic system by using a novel fusion tag based on a Vip3 protein signal peptide sequence or a partial sequence thereof to guide the expression of the target protein. The fusion gene formed by the fusion tag and the coding gene of the target protein and the escherichia coli molecular chaperone Trigger gene or homologous genes of the escherichia coli molecular chaperone Trigger gene in other organisms are co-expressed in the same prokaryotic expression system, so that the expression quantity and the solubility of the target protein can be enhanced. The invention has the importance that a novel fusion tag is invented, the expression quantity of target proteins can be obviously enhanced, meanwhile, the tag can interact with known specific molecular chaperones, so that the solubility of the target proteins can be predictably enhanced on the premise of ensuring the expression quantity, the traditional scheme of enhancing the solubility of unknown target proteins can be determined only by virtue of one-by-one attempts of different molecular chaperones, and the efficiency is greatly improved.

Description

Method for simultaneously enhancing expression quantity and solubility of target protein in prokaryotic system

Technical Field

The invention relates to the technical field of biology, in particular to a method for simultaneously enhancing the expression quantity and the solubility of target proteins in a prokaryotic system.

Background

In the expression of recombinant proteins of interest using prokaryotic systems, to increase the solubility of the protein of interest, inclusion body formation is prevented and may be regulated using a variety of soluble tags, such as MBP (maltose-binding protein) (di Guanetal 1988; kapush and Waugh 1999) tag, GST (glutethione-S-transferase) (Smith and Johnson, 1988) tag, SUMO (small ubiquitin related modifier) (button et al 2005; marblestone et al 2006), nusA (N-utilization substance) (Davis et al 1999) tag, trx (Thioredoxin) (Lavallie et al 1993) and the like. After the label is connected to the N end of the target protein, the solubility of the target protein can be increased, so that the target protein is easier to form a correct spatial structure. Some tags also increase the expression level of the target protein. Of course, the positive effect difference of different labels on the expression of different target proteins is large, and the fact that a specific fusion label is effective in promoting the dissolution of which protein cannot be prejudged at present is proved by experiments, so that the development of a novel label has good application prospect.

The bacterial secretion Signal Peptide (SP) is capable of transporting a protein of interest to the periplasmic space of a gram-negative bacterium or to the outside of the cell of the bacterium. When the target protein is expressed, the signal peptide can prevent cytotoxicity caused by excessive accumulation of the target protein, especially toxic protein, in cells or prevent unexpected degradation of recombinant target protein by intracellular protease, thereby improving the expression level of the target protein. In addition, part of the target protein can be folded better in the periplasmic space or outside the bacteria, so that the secretion signal peptide is used as a fusion tag, and the soluble expression of the part of the recombinant target protein can be improved.

Most secretion signal peptides have 3 classical regions, from N-terminal to C-terminal, N-region in turn, with stronger hydrophilicity and net positive charge; h-region, the hydrophobicity is stronger; c-region, the hydrophobicity is reduced. Most of the secretory signal peptide is removed by the corresponding signal peptidase during or after transport. The present invention uses a novel fusion tag, namely the secretion signal peptide (Vsp) of the secreted insecticidal protein Vip3 (Estruch et al 1996; crickmore et al 2020) from Bacillus thuringiensis (Bacillus thuringiensis). The biggest characteristic of the signal peptide is that the recognition site of the secretion signal peptide is not available, so that the Vip3 protein still carries the secretion signal peptide after transmembrane transport. This feature allows for the ability to affect the solubility and spatial structure of recombinant proteins of interest after the polypeptide has been translated.

The invention uses Vsp (Doss et al 2002; chen et al 2003; rang et al 2005; chakroun et al 2016) or a partial sequence thereof as a fusion tag to guide a target protein, thereby remarkably increasing the expression level. In addition, the local sequences of Vsp, i.e., N-region (VspN), H-region (VspH), and C-region (VspC), and various combinations thereof, such as VspNH, vspNC, vspHC, can increase the expression level of the recombinant protein of interest. Among them, vspN itself has a relatively high hydrophilicity and net positive charge, and thus, can also promote the solubility of a protein of interest. However, since VspH and VspC are relatively hydrophobic, the presence of these two regions, either separately or simultaneously, has a negative effect on the solubility of the target protein. However, the present invention has found that Vsp or its partial sequence can be specifically recognized by a chaperone Trigger (TF) (De Geyter et al 2020), i.e., the solubility can be significantly or completely restored by coexpression of the Trigger with a target protein containing a Vsp signal peptide or its partial sequence.

Molecular chaperones such as Trigger factor, dnaK-dnaJ-grpE, groES-groEL are often co-expressed with recombinant target protein in a prokaryotic expression system, thereby improving the solubility of the target protein. However, it is not yet possible to predict which target protein interacts with a specific chaperone and produces a lytic effect. Conventionally, attempts have been made one by one, such as Chaperone Plasmid Set of the company Takara, japan, to provide a coexpression scheme of a plurality of chaperones, so that users have a one-by-one effect, and the work efficiency has been lowered.

The invention has the importance of providing a novel fusion tag for improving the expression quantity of target protein in a prokaryotic system, and simultaneously defining the specific effect of a Trigger Factor on the recombinant target protein guided by the tag, so that a user can predict the expression quantity of the target protein, and simultaneously predict the solubility of a final product, thereby greatly improving the working efficiency.

Disclosure of Invention

The invention solves the technical problem of providing a method for simultaneously enhancing the expression quantity and the solubility of a target protein in a prokaryotic system, and using a polynucleotide sequence encoding a Vip3 protein signal peptide sequence or a partial sequence thereof or mutants of the polypeptide sequences or corresponding artificial simulation polypeptide sequences as a novel fusion tag to form a fusion gene with the polynucleotide sequence encoding the target protein. The expression level of the fusion genes in a prokaryotic system is obviously improved. The fusion gene and escherichia coli molecular chaperone Trigger gene or homologous genes thereof in other organisms are co-expressed in the same prokaryotic expression system, so that the expression quantity and the solubility of target proteins can be enhanced.

The technical scheme of the invention is as follows:

firstly, the method for simultaneously enhancing the expression quantity and the solubility of the target protein in a prokaryotic system is provided by the invention, wherein a novel fusion tag is used for forming a fusion protein with the target protein, and comprises a Vip3 protein signal peptide sequence or a partial sequence thereof or mutants of the polypeptide sequences or corresponding artificial simulation polypeptide sequences; the coding gene of the fusion protein and the escherichia coli molecular chaperone Trigger gene or homologous genes thereof in other organisms are co-expressed in the same prokaryotic system, so that the expression quantity and the solubility of the target protein are realized.

Furthermore, in the scheme, the amino acid sequence of the signal peptide sequence of the Vip3 protein is shown as SEQ ID NO. 1-26, the amino acid sequence of the partial sequence of the signal peptide sequence of the Vip3 protein is shown as SEQ ID NO. 27-32, and the polynucleotide sequence of the coding polypeptide sequence related to the invention can be deduced according to the genetic code. The preference of codon usage can also be adjusted according to the type of host cell. Knowledge of the genetic code and the preferences of codon usage is fundamental in the field of biology, and thus polynucleotide sequences can be prepared by conventional molecular biological methods or chemical synthesis methods well known in the art.

Further, in the above scheme, the key mutations or mimetic principles of the Vip3 protein signal peptide sequence or the mutant of its partial sequence or the corresponding artificial mimetic polypeptide sequence are: the first is that lysine and arginine in the original polypeptide sequence can be interchanged, but the total number of the two basic amino acids can only be increased and cannot be reduced, and the second is that amino acids at other positions except for lysine and arginine at corresponding positions in the original sequence can be replaced by basic amino acids or other R-group non-hydrophobic amino acids. Other references herein refer to amino acids at positions other than the lysine and arginine at the corresponding positions in the original sequence.

Further, in the scheme, the polypeptide sequence of the escherichia coli molecular chaperone Trigger is shown as SEQ ID NO. 33. When the polynucleotide encoding the protein or the mutant or the homologous sequence thereof in other species and the encoding gene of the fusion protein are co-expressed in the same system, the Vip3 protein signal peptide sequence or the partial sequence thereof or the mutant of the polypeptide sequences or the corresponding artificial simulation polypeptide sequences can be identified and interacted with each other, so that the predictability is ensured while the expression quantity is ensured.

Further, in the above-described scheme, in the fusion protein formed by the Vip3 protein signal peptide sequence or a partial sequence thereof or a mutant of the polypeptide sequences or the corresponding artificial mimetic polypeptide sequences and the target protein, the Vip3 protein signal peptide sequence or a partial sequence thereof or a mutant of the polypeptide sequences or the corresponding artificial mimetic polypeptide sequences are located at the N-terminal of the target protein, the Vip3 protein signal peptide sequence or a partial sequence thereof or a mutant of the polypeptide sequences or the corresponding artificial mimetic polypeptide sequences and the target protein sequences may be directly connected, or a spacer sequence may be present.

Further, in the above-described embodiment, the spacer sequence in the fusion protein is an amino acid sequence encoded by a polynucleotide sequence of a restriction enzyme recognition site; other common fusion tag sequences, or linking sequences formed by the concatenation of small molecule amino acids, or coding polynucleotide sequences such as protease recognition sequences, intein sequences, etc. are also possible.

Further, in the above-described scheme, the expression level of the fusion protein is significantly increased compared with the expression level of the target protein which does not contain the Vip3 protein signal peptide sequence or a partial sequence thereof or mutants of these polypeptide sequences or corresponding artificial mimetic polypeptide sequences, but the solubility of the fusion protein is somewhat different depending on the different target proteins.

Further, in the above-described scheme, the polynucleotide sequences encoding the fusion protein and chaperone may be expressed under the control of known regulatory sequences in molecular biology such as promoters, enhancers, terminators, operators, 2A polypeptides, etc.

Further, in the above-described scheme, the Vip3 protein signal peptide sequence or a partial sequence thereof or a mutant of these polypeptide sequences or a polynucleotide sequence of the corresponding artificial mimetic polypeptide sequence can interact with the sequence of the chaperone Trigger or a mutant sequence thereof or a homologous sequence thereof in other species, thereby enhancing the expression of the target protein in the prokaryotic system and/or improving the solubility of the expressed protein and/or promoting the correct formation of the spatial structure of the target protein.

Further, in the above scheme, the polynucleotide sequences encoding the fusion protein and the chaperone may be placed in the same DNA molecule or DNA vector at the same time, or may be placed in different DNA molecules or DNA vectors, so that expression is regulated by corresponding regulatory sequences, such as the 3' end of a promoter for promoting expression of the target gene, or directly replace the existing gene sequence in the vector. When the polynucleotide sequences encoding the fusion protein and chaperones are placed in the same DNA molecule or DNA vector, they must not be translated in one reading frame each independently.

Further, in the above scheme, the co-expression means that two genes are expressed in the same system, but the transcription and translation processes may or may not be performed simultaneously.

Still further, the DNA vector is a natural or artificial plasmid vector, a natural or artificial phage genome, a natural or artificial microbial genome, a natural or artificial eukaryotic genome, other natural or artificial DNA fragments.

Further, in the above-described embodiments, the target protein includes a protein or polypeptide that can be expressed in a prokaryotic system by biological techniques, including a protein or polypeptide that is native to the host system, and also includes a protein or polypeptide that is not native to the host system.

Further, in the above-described embodiments, the prokaryotic system includes a target protein expression system based on a living prokaryotic cell, and also includes an in vitro protein expression system constructed using all or a part of components required for protein synthesis in a prokaryotic cell.

The beneficial effects of the invention are as follows: the invention firstly provides a novel fusion tag which can enhance the expression quantity of target protein in a prokaryotic system. Such novel fusion tags include Vip3 protein signal peptide sequences or partial sequences thereof or mutants of these polypeptide sequences or corresponding artificial mimetic polypeptide sequences. These sequences can increase the expression level of the target protein, and partial sequences can promote the solubility of the target protein. More importantly, when the fusion target protein containing the sequences is co-expressed with an escherichia coli molecular chaperone Trigger or a mutant sequence thereof or a homologous sequence thereof in other species, the solubility of the target protein can be obviously improved in predictability, even completely dissolved. Therefore, the novel fusion tag can be used as a powerful supplement of the existing protein expression tag or expression method, and meanwhile, the novel fusion tag can be combined with an escherichia coli molecular chaperone Trigger or a mutant sequence thereof or a homologous sequence thereof in other species, so that a clear scheme for improving the expression quantity and the solubility of target proteins can be provided.

The inventor also invents a method for improving the expression quantity and the solubility of target proteins by using a secretion signal peptide of a secretion insecticidal protein Cry1I of bacillus thuringiensis (Bacillus thuringiensis) as a novel fusion tag. The invention has the common point with the invention that the invention is the research and the utilization of the expansion application of the separated signal peptide. But there are significant differences between the two inventions. First, cry1I and Vip3 proteins are a family of disparate insecticidal proteins, differing in amino acid sequence, spatial structure, and receptor proteins within the target insect body; secondly, the biggest point of the Cry1I secretion signal peptide as a fusion tag for enhancing the expression quantity and the solubility of the target protein is that the Cry1I secretion signal peptide can not be used as the secretion signal peptide for efficiently transferring the target protein through a membrane, but is reserved in cytoplasm of escherichia coli, and the promotion effect of the signal peptide on the expression quantity of the protein is very remarkable. Third, the Vip3 secretion signal peptide of the present invention is capable of transporting a protein of interest, unlike the Cry1I secretion signal peptide. Fourth, the present invention has studied a plurality of partial sequences of Vip3 secretion signal peptide, and found that these sequences can significantly increase the expression level of the target protein, and that partial sequences can even increase the solubility of the target protein. Therefore, the novel fusion tag series related to the invention can provide more choices for users. Fifth, and more important, the invention directly screens the combination scheme of the Vip3 series fusion tag and the specific molecular chaperone Trigger. The scheme has the greatest advantages that the novel fusion tag is utilized to ensure the expression quantity of the target protein, meanwhile, clear soluble expectation is provided for a user, and the traditional scheme of high manpower, material resources and financial resource consumption of the available scheme can be determined only by means of different molecular chaperone combination attempts is avoided. In the invention of Cry1I secretion signal peptide, the corresponding molecular chaperones are not clear.

Drawings

FIG. 1 is a schematic diagram of expression vectors p28aD-eGFP (A) and p28aD-VeGFP (B) in example 2. Wherein, in example 6, modified regions of genes encoding partial sequences of different Vip3 secretion signal peptides are marked in panel B.

FIG. 2 is a graph showing comparison of eGFP and VeGFP expression in E.coli BL21 (DE 3) star strain in example 3 and a graph showing detection of expression of the VeGFP mutant fusion protein in example 6; wherein, lane "1" represents eGFP; lane "2" represents VeGFP; lane "3" represents VNeGFP; lane "4" represents VHeGFP; lane "5" represents VCeGFP; lane "6" represents VNHeGFP; lane "7" represents VNCeGFP; lane "8" represents vhcelgfp; lane "M" represents standard protein molecular weight; lanes "-" represent negative control (prepared with BL21 (DE 3) star strain containing pET28a plasmid); the arrows indicate the bands of interest for eGFP and VeGFP, respectively, and mutants thereof.

FIG. 3 is a schematic diagram of a vector in which the VeGFP encoding gene and the Trigger Factor gene are co-expressed. The co-expression vector of the VeGFP encoding gene and the encoding genes of other 5 chaperones or corresponding mutants in example 4 only replaces the Trigger Factor gene therein (labeled as a); in example 6, the vector for coexpression of the VeGFP mutant-encoding gene and other Trigger Factor genes was replaced by only the Vip3A secretion signal peptide portion (labeled B).

FIG. 4 is a graph showing the solubility of the different chaperones or corresponding mutants in example 5 for the expression of VeGFP in E.coli BL21 (DE 3) star strain; wherein, lane "T" represents the total protein amount (cell ultrasound lysate); lane "S" represents the soluble fraction (supernatant obtained by centrifugation after cell sonication); lane "P" represents insoluble fraction (pellet fraction obtained by centrifugation after cell sonication); lane "M" represents the standard protein molecular weight, and "pET28aDel" represents the negative control (prepared with BL21 (DE 3) star strain containing pET28a plasmid); white arrows indicate the destination bands of VeGFP and black arrows indicate the destination bands of the different chaperones, respectively.

FIG. 5 is a diagram showing the hydrophobicity profile of the N-terminal signal peptide amino acid sequence of the Vip3Aa protein described in example 6 and its comparison with the signal peptides of the three common secreted proteins. The N-terminal signal peptide of the Vip3Aa protein; MBP protein N-terminal signal peptide; a PelB protein N-terminal signal peptide; tora protein N-terminal signal peptide; E.Vip3 protein N region (N-region), H region (N-region) and C region (C-region) prediction scheme.

FIG. 6 is a soluble analysis chart (A) of the independent expression of 6 VeGFP mutants in example 6 and a soluble expression analysis chart of 6 VeGFP mutants when co-expressed with a chaperone Trigger in example 7. Wherein, lane "T" represents the total protein amount (cell ultrasound lysate); lane "S" represents the soluble fraction (supernatant obtained by centrifugation after cell sonication); lane "P" represents insoluble fraction (pellet fraction obtained by centrifugation after cell sonication); lane "M" represents the standard protein molecular weight, and "pET28aDel" represents the negative control (prepared with BL21 (DE 3) star strain containing pET28a plasmid); white arrows indicate the destination bands of VeGFP and black arrows indicate the destination bands of the different chaperones, respectively.

FIG. 7 is a comparison of the quantitative analysis of the solubility and the difference in significance of 6 VeGFP mutants in example 6 and example 7. Where "TF-" represents the percentage of soluble portion of the protein of interest in total protein of interest when 6 VeGFP mutants were expressed alone (example 6), and "TF+" represents the percentage of soluble portion of the protein of interest in total protein of 6 VeGFP mutants when co-expressed with a chaperone Trigger (example 7). The number of repetitions per protein is 3 or more.

Detailed Description

The present invention will be described in detail by the following examples for better understanding of the technical aspects of the present invention, but the scope of the present invention is not limited thereto.

Example 1: construction of fusion protein VeGFP

This example illustrates a fusion protein VeGFP in which the N-terminal signal peptide of the naturally occurring protein Vip3Aa (SEQ ID NO: 1) is spliced with the eGFP protein.

The VeGFP protein is characterized in that: the N-terminal signal peptide sequence of Vip3Aa is positioned at the N end of eGFP protein; there are NO additional redundant amino acids between the N-terminal signal peptide of Vip3Aa (SEQ ID NO: 1) and the amino acid sequence of the eGFP protein. The coding polynucleotide sequence for the VeGFP protein can be obtained from at least three pathways: 1) The polynucleotide sequences of the N-terminal signal peptide encoding Vip3Aa and the eGFP protein amino acid sequence are respectively obtained by polymerase chain reaction (PCR reaction), and then the two are spliced together by a method of overlapping PCR (Horton, R.M. et al BioTechniques, 1990) to form a complete fusion gene; 2) The polynucleotide sequences of the N-terminal signal peptide encoding Vip3Aa and the eGFP protein amino acid sequence are respectively obtained by polymerase chain reaction (PCR reaction), and then are spliced into a complete fusion gene by a recombinant cloning method; 3) The polynucleotide sequences encoding the two proteins are directly spliced by artificial synthesis. All three methods are conventional molecular biological methods or conventional synthetic methods well known in the art.

Example 2: construction of prokaryotic cell expression vector p28aD-VeGFP of fusion protein VeGFP

This example illustrates a prokaryotic expression vector for the fusion protein described in example 1 herein. First, bamHI (5 ') and SacI (3') sites were added to both ends of the VeGFP gene, respectively. The gene was then inserted into the pET28aDel vector (Gao et al 2011) at the corresponding site to form a p28aD-VeGFP expression vector (FIG. 1). As a control, bamHI (5 ') and SacI (3') sites were added to both ends of the eGFP gene, respectively, to form a p28aD-eGFP expression vector (FIG. 1). Two plasmids were transformed into E.coli BL21 (DE 3) star strain to obtain BL28-VeGFP and BL28-eGFP strains, respectively.

Example 3: expression of VeGFP protein and eGFP protein and comparison

BL28-VeGFP and BL28-eGFP strains were streaked on solid LB medium (Luria-Bertani medium) containing 50. Mu.g/mL kanamycin, respectively, and cultured overnight at 37 ℃. 6 individual clones were selected for each strain, individually picked and inoculated into liquid LB medium containing 50. Mu.g/mL kanamycin, and shake cultured (37 ℃,200 rpm) to OD600 = 0.8. IPTG (isopropylβ -D-1-thiohinging) was added to the medium at a final concentration of 1 mmol/L. Expression was induced for 16 hours at 16 ℃. After the induction expression is finished, each tube of bacterial liquid is sucked into 500 mu L of a new 1.5mL centrifuge tube, and the bacterial cells are collected by centrifugation at 12000rpm for 5 min. 200. Mu.L of PBS buffer (137mM NaCl,2.7mM KCl,10mM Na) ₂ HPO ₄ ,and 2mM KH ₂ PO ₄ pH 7.4) and 50. Mu.L of 5 Xprotein loading buffer (250 mM Tris (pH 6.8), 10% (w/v) SDS,0.5% (w/v) BPB,50% (v/v) glycerol, 5% (v/v) beta-mercaptoethanol) were added and mixed. After boiling for 10min, centrifugation was carried out at 12000rpm for 5min, 10. Mu.L of the supernatant was spotted, and SDS-PAGE was performed to analyze the protein expression. As a result, as shown in FIG. 2, the expression level of eGFP was low, and the molecular weight was very close to that of the host cell self-protein, whereas the expression level of VeGFP was significantly higher than that of eGFP.

Example 4: construction of different VeGFP molecular chaperone expression vectors

Co-expression of the coding genes of VeGFP and E.coli chaperones Trigger (SEQ ID NO. 33), secB (SEQ ID NO. 34), secB7577 (SEQ ID NO. 35), secB142 (SEQ ID NO. 36), secB142_7577 (SEQ ID NO. 37) or SecA (SEQ ID NO. 38) was achieved using 6 expression vectors p28aD-VeGFP-SecA, p28aD-VeGFP-SecB7577, p28aD-VeGFP-SecB142, p28aD-VeGFP-142_7577 and p28aD-VeGFP-TF, respectively. The sequence construction is realized by a mode of artificial synthesis. The main structure is that after the restriction enzyme SacI site at the 3' end of the VeGFP gene is added, kpnI restriction enzyme recognition sequence is added, then bacillus thuringiensis CsaA gene promoter sequence is added, then coding genes of escherichia coli molecular chaperone TF (SEQ ID NO. 33), secB (SEQ ID NO. 34), secB7577 (SEQ ID NO. 35), secB142 (SEQ ID NO. 36), secB142_7577 (SEQ ID NO. 37) or SecA (SEQ ID NO. 38) are respectively and independently connected, and 6 VeGFP and the chaperones are respectively obtained to form a co-expression frame. The end of each co-expression frame was regulated for transcription termination by the T7 terminator sequence of the pET28aDel vector (fig. 3). The 6 plasmids were transformed into E.coli BL21 (DE 3) star strains, respectively, to obtain BL28-VeGFP-SecA, BL28-VeGFP-SecB7577, BL28-VeGFP-SecB142, BL28-VeGFP-SecB142_7577 and BL28-VeGFP-TF strains.

Example 5: comparing the effect of different chaperones on soluble expression of VeGFP fusion proteins

BL28-eGFP, BL28-VeGFP-SecA, BL28-VeGFP-SecB7577, BL28-VeGFP-SecB142, BL28-VeGFP-SecB142_7577 and BL28-VeGFP-TF strains were streaked on solid LB medium (Luria-Bertani medium) containing 50. Mu.g/mL kanamycin, respectively, and cultured overnight at 37 ℃.3 single clones were selected for each strain, and were individually picked up and inoculated into liquid LB medium containing 50. Mu.g/mL kanamycin overnight for culture, and after the completion of the culture, 50. Mu.L of the bacterial liquid was sequentially inoculated into a conical flask containing 50mL of liquid LB medium for shaking culture (37 ℃,200 rpm) until OD600 = 0.8. IPTG (isopropylβ -D-1-thiohinging) was added to the medium at a final concentration of 1 mmol/L. Expression was induced for 16 hours at 16 ℃. After the induction was completed, 50mL of the bacterial liquid contained in each conical flask was collected into a 50mL centrifuge tube, and the bacterial cells were collected by centrifugation at 12000rpm for 10min in a centrifuge at 4 ℃. With 20ml PBS buffer (137mM NaCl,2.7mM KCl,10mM Na) ₂ HPO ₄ ,and 2mM KH ₂ PO ₄ pH 7.4) resuspension of cells, crushing for 30min under ice bath condition by using a cell ultrasonic crusher, taking 200 mu L to two new 1.5ml centrifuge tubes in turn after crushing of each tube of cell resuspension, centrifuging at 12000rpm of one centrifuge tube for 5min, separating soluble part and insoluble part, adding 50 mu L of 5 Xprotein loading buffer (250 mM Tris (pH 6.8), 10% (w/v) SDS,0.5% (w/v) BPB,50% (v/v) glycerol and 5% (v/v) beta-mercaptoethanol), and uniformly mixing. After boiling for 10min, centrifugation was carried out at 12000rpm for 5min, 10. Mu.L of the supernatant was spotted, and SDS-PAGE was performed to analyze the protein expression. The results are shown in FIG. 4, 6 chaperones or partitionsThe chaperone mutants are successfully co-expressed with VeGFP, but only Trigger factor can obviously promote the solubility of VeGFP, and other chaperones in 5 have no corresponding effect.

Example 6: construction of fusion tag vectors for different VeGFP mutants and comparison of soluble expression

The domains were found to be divided into positively charged N-regions (from M1 to R11, N-regions) followed by hydrophobic residues (H region, from A12 to F20, N-region) and C region (the remaining region, C-region) by sequence alignment and hydrophobicity prediction of the N-terminal signal peptide of the Vip3Aa protein (FIG. 5). The three segments were combined singly or in pairs to form six different Vip3Aa signal peptide mutant sequences of VN (SEQ ID No. 27), VH (SEQ ID No. 28), VC (SEQ ID No. 29), VNH (SEQ ID No. 30), VNC (SEQ ID No. 31) and VHC (SEQ ID No. 32), and the encoding genes of the mutant sequences were fused to eGFP genes to obtain 6 different VeGFP mutant fusion tag genes VNeGFP, VHeGFP, VCeGFP, VNHeGFP, VNCeGFP and vhcelfp genes. These fusion tag genes were inserted into BamHI (5 ') and SacI (3') sites of pET28aDel vector, respectively, to obtain p28aD-VNEGFP, p28aD-VHeGFP, p28aD-VCeGFP, p28aD-VNEGFP and p28aD-VHCEGFP expression vectors. Six plasmids were transformed into E.coli BL21 (DE 3) star strains, respectively, to obtain BL28-VNEGFP, BL28-VHeGFP, BL28-VCeGFP, BL28-VNEGFP and BL28-VHCEGFP strains.

BL28-VNEGFP, BL28-VHeGFP, BL28-VCeGFP, BL28-VNEGFP and BL28-VHCEGFP strains were streaked out in solid LB medium (Luria-Bertani medium) containing 50. Mu.g/mL kanamycin, respectively, and cultured overnight at 37 ℃.3 single clones were selected for each strain, and were individually picked up and inoculated into liquid LB medium containing 50. Mu.g/mL kanamycin overnight for culture, and after the completion of the culture, 50. Mu.L of the bacterial liquid was sequentially inoculated into a conical flask containing 50mL of liquid LB medium for shaking culture (37 ℃,200 rpm) until OD600 = 0.8. IPTG (isopropylβ -D-1-thiohinging) was added to the medium at a final concentration of 1 mmol/L. Expression was induced for 16 hours at 16 ℃. After the induction expression was completed, 50mL of the bacterial liquid contained in each conical flask was collected into a 50mL centrifuge tube, and the bacterial liquid was collected by centrifugation at 12000rpm for 10min in a centrifuge at 4 ℃And (3) cells. With 20PBS buffer (137mM NaCl,2.7mM KCl,10mM Na) ₂ HPO ₄ ,and 2mM KH ₂ PO ₄ pH 7.4) resuspension of cells, crushing for 30min under ice bath condition by using a cell ultrasonic crusher, taking 200 mu L to two new 1.5ml centrifuge tubes in turn after crushing of each tube of cell resuspension, centrifuging at 12000rpm of one centrifuge tube for 5min, separating soluble part and insoluble part, adding 50 mu L of 5 Xprotein loading buffer (250 mM Tris (pH 6.8), 10% (w/v) SDS,0.5% (w/v) BPB,50% (v/v) glycerol and 5% (v/v) beta-mercaptoethanol), and uniformly mixing. After boiling for 10min, centrifugation was carried out at 12000rpm for 5min, 10. Mu.L of the supernatant was spotted, and SDS-PAGE was performed to analyze the protein expression. As shown in FIG. 6A, the expression level of each of the 6 VeGFP mutants was higher than that of eGFP, and the solubility of each of the VeGFP mutants was higher than that of VeGFP.

Example 7: construction of different VeGFP mutant fusion proteins and comparison of solubility of target proteins with chaperone Co-expression vectors

The genes encoding the different VeGFP mutant fusion proteins were ligated into BamH I (5 ') and Sac I (3') sites of the p28aD-VeGFP-TF vectors of example 4, respectively, to obtain p28aD-VNEGFP-TF, p28aD-VHeGFP-TF, p28aD-VCeGFP-TF, p28aD-VNEGFP-TF, p28aD-VNCEGFP-TF and p28aD-VHCeGFP-TF expression vectors (FIG. 3). Six plasmids were transformed into E.coli BL21 (DE 3) star strains, respectively, to obtain BL28-VNEGFP-TF, BL28-VHeGFP-TF, BL28-VCeGFP-TF, BL28-VNEGFP-TF and BL28-VHCEGFP-TF strains.

BL28-VNEGFP-TF, BL28-VHeGFP-TF, BL28-VCeGFP-TF, BL28-VNEGFP-TF and BL28-VHCEGFP-TF strains were streaked on solid LB medium (Luria-Bertani medium) containing 50. Mu.g/mL kanamycin, respectively, and cultured overnight at 37 ℃.3 single clones were selected for each strain, and were individually picked up and inoculated into liquid LB medium containing 50. Mu.g/mL kanamycin overnight for culture, and after the completion of the culture, 50. Mu.L of the bacterial liquid was sequentially inoculated into a conical flask containing 50mL of liquid LB medium for shaking culture (37 ℃,200 rpm) until OD600 = 0.8. IPTG (isopropylβ -D-1-thiohinging) was added to the medium at a final concentration of 1 mmol/L. Expression was induced for 16 hours at 16 ℃. After the induction of expression is completed50mL of the bacterial liquid contained in each conical flask was collected into a 50mL centrifuge tube, and centrifuged at 12000rpm in a centrifuge at 4℃for 10min to collect bacterial cells. With 20PBS buffer (137mM NaCl,2.7mM KCl,10mM Na) ₂ HPO ₄ ,and 2mM KH ₂ PO ₄ pH 7.4) resuspension of cells, crushing for 30min under ice bath condition by using a cell ultrasonic crusher, taking 200 mu L to two new 1.5ml centrifuge tubes in turn after crushing of each tube of cell resuspension, centrifuging at 12000rpm of one centrifuge tube for 5min, separating soluble part and insoluble part, adding 50 mu L of 5 Xprotein loading buffer (250 mM Tris (pH 6.8), 10% (w/v) SDS,0.5% (w/v) BPB,50% (v/v) glycerol and 5% (v/v) beta-mercaptoethanol), and uniformly mixing. After boiling for 10min, centrifugation was carried out at 12000rpm for 5min, 10. Mu.L of the supernatant was spotted, and SDS-PAGE was performed to analyze the protein expression. The expression results are shown in FIG. 6B. Comparison of the expression results of 6 VeGFP mutants expressed alone (fig. 6A) and 6 VeGFP mutants co-expressed with chaperone TF (fig. 6B) revealed that the solubility of the protein of interest was improved. The quantitative analysis results show that the dissolution-promoting effect of the water-soluble polymer reaches the extremely remarkable level (P<0.01, n.gtoreq.3) (FIG. 7).

The specific sequences of SEQ ID NO.1-SEQ ID NO.38 are shown below:

SEQ ID NO.1：MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.2：MNKNNTKLSTRALPSFIDYFNGVYGFATGIKDI

SEQ ID NO.3：MNKNNTKLNTRALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.4：MNKNNTKLNARALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.5：MNKNNTKLSARALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.6：MNMNKNNTKLSARALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.7：MNKNNTKLSTRALPSFIDYFNGIYGFTTGIKDI

SEQ ID NO.8：MNKNNTKLSTRALPGFIDYFNGIYGFATGIKDI

SEQ ID NO.9：MTKNNTKLSTRALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.10：MNMNNTKLNARALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.11：MNNTKLNARALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.12：MNMNNTKLSARALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.13：MNMNNTKLSARALPSLIDYFNGIYGFATGIKDI

SEQ ID NO.14：MNMNNTKLSTRALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.15：MNMNNTKLSTRALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.16：MNMNNAKLNARALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.17：MNMNNTKLNARALPSFIDYFNGIYGFAIGIKDI

SEQ ID NO.18：MNMNNAKLNARALPSFIDYFNGIYGFAIGIKDI

SEQ ID NO.19：MNKNNTKLNARALPSFIDYFNGIYGFAIGIKDI

SEQ ID NO.20：

MVQKWMQRMIIVDNNKLNVRALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.21：MQKNNKLSVKALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.22：MNKNNTKLSHVVVISFIDYFNGIYGFATGIKDI

SEQ ID NO.23：MNKNNTKLSNVNELSSLSDYFNGIYGFATGIKDI

SEQ ID NO.24：MNKNNTKLSKNELSRLSDYFNGIYGFATGIKDI

SEQ ID NO.25：MNKNNTKLSKNELSRLSDYFNGIYGFATGIKDI

SEQ ID NO.26：MNKNNTKLSNFRCLYLVEYFNGIYGFATGIKDI

SEQ ID NO.27：MNKNNTKLSTR

SEQ ID NO.28：MALPSFIDYF

SEQ ID NO.29：MNGIYGFATGIKDI

SEQ ID NO.30：MNKNNTKLSTRALPSFIDYF

SEQ ID NO.31：MNKNNTKLSTRNGIYGFATGIKDI

SEQ ID NO.32：MALPSFIDYFNGIYGFATGIKDI

SEQ ID NO.33：

MSTKWEKLEGNVGVLTIEVDAKEVNNSIDAAFKKVVKTINVPGFRKGKMPRPLFEQRFGIESLYQDALDIILPKAYGEAIEEAGIFPVDHPEIDIEKFEKNANLIFTAKVTVKPEVKLGEYKGLAVEKVETTVTDEDVENELKSLQERQAELVVKEEGTVENGDTAVIDFEGFVDGEAFEGGKGENYSLAIGSGTFIPGFEEQVIGLKSGESKDVEVSFPEEYHAAELAGKPATFKVTIHEIKTKELPELNDEFAKEADEAVATLDELKAKLRTNLEEGKKHEAEHKVRDEVVELAAANAEIEIPEAMINTELDRMVREFEQRLSQQGMNLELYYQFTGTDADKLKEQMKEDAQKRVRINLVLEAIIEAENIEVTEEEVTAEVEKMAEMYGMPVDAIKQALGSVDALAEDLKVRKAVDFLVENAA

SEQ ID NO.34：

MSEQNNTEMTFQIQRIYTKDISFEAPNAPHVFQKDWQPEVKLDLDTASSQLADDVYEVVLRVTVTASLGEETAFLCEVQQGGIFSIAGIEGTQMAHCLGAYCPNILFPYARECITSMVSRGTFPQLNLAPVNFDALFMNYLQQQAGEGTEEHQDA

SEQ ID NO.35：

MSEQNNTEMTFQIQRIYTKDISFEAPNAPHVFQKDWQPEVKLDLDTASSQLADDVYEVVLRVTVTASLGEETAFQCVVQQGGIFSIAGIEGTQMAHCLGAYCPNILFPYARECITSMVSRGTFPQLNLAPVNFDALFMNYLQQQAGEGTEEHQDA

SEQ ID NO.36：

MSEQNNTEMTFQIQRIYTKDISFEAPNAPHVFQKDWQPEVKLDLDTASSQLADDVYEVVLRVTVTASLGEETAFLCEVQQGGIFSIAGIEGTQMAHCLGAYCPNILFPYARECITSMVSRGTFPQLNLAPVNFDALFMNYLQ

SEQ ID NO.37：

MSEQNNTEMTFQIQRIYTKDISFEAPNAPHVFQKDWQPEVKLDLDTASSQLADDVYEVVLRVTVTASLGEETAFQCVVQQGGIFSIAGIEGTQMAHCLGAYCPNILFPYARECITSMVSRGTFPQLNLAPVNFDALFMNYLQ

SEQ ID NO.38：

MIGILKKVFDVNQRQIKRMQKTVEQIDALESSIKPLTDEQLKGKTLEFKERLTKGETVDDLLPEAFAVVREAATRVLGMRPYGVQLMGGIALHEGNISEMKTGEGKTLTSTLPVYLNALTGKGVHVVTVNEYLAQRDASEMGQLHEFLGLTVGINLNSMSREEKQEAYAADITYSTNNELGFDYLRDNMVLYKEQCVQRPLHFAIIDEVDSILVDEARTPLIISGQAQKSTELYMFANAFVRTLENEKDYSFDVKTKNVMLTEDGITKAEKAFHIENLFDLKHVALLHHINQGLRAHVVMHRDTDYVVQEGEIVIVDQFTGRLMKGRRYSEGLHQAIEAKEGVEIQNESMTLATITFQNYFRMYEKLSGMTGTAKTEEEEFRNIYNMNVIVIPTNKPIIRDDRADLIFKSMEGKFNAVVEDIVNRHKKGQPVLVGTVAIETSELISKMLTRKGVRHNILNAKNHAREADIIAEAGIKGAVTIATNMAGRGTDIKLGDDVKNVGLAVIGTERHESRRIDNQLRGRAGRQGDPGVTQFYLSMEDELMRRFGSDNMKAMMDRLGMDDSQPIESKMVSRAVESAQKRVEGNNYDARKQLLQYDDVLRQQREVIYKQRQEVMESDNLRGIIEGMMKSTVERAVALHTQEEIEEDWNIKGLVDYLNTNLLQDGDVKEEELRRLAPEEMSEPIIAKLIERYNEKEKLMPEEQMREFEKVVVFRVVDTKWTEHIDAMDHLREGIHLRAYGQIDPLREYQMEGFAMFESMVASIEEEISRYIMKAEIEQNLERQEVVQGEAVHPSSDGEEAKKKPVVKGDQVGRNDLCKCGSGKKYKNCCGIVQ

finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Sequence listing

<110> Shanxi university of agriculture

<120> method for simultaneously enhancing expression amount and solubility of target protein in prokaryotic system

<130> none of

<141> 2022-01-28

<160> 38

<170> SIPOSequenceListing 1.0

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Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 2

<211> 32

<212> PRT

<213> Abedus herberti

<300>

<313> (1)..(32)

<400> 2

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Val Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

<210> 3

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 3

Met Asn Lys Asn Asn Thr Lys Leu Asn Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 4

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 4

Met Asn Lys Asn Asn Thr Lys Leu Asn Ala Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 5

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 5

Met Asn Lys Asn Asn Thr Lys Leu Ser Ala Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 6

<211> 35

<212> PRT

<213> A

<300>

<313> (1)..(35)

<400> 6

Met Asn Met Asn Lys Asn Asn Thr Lys Leu Ser Ala Arg Ala Leu Pro

1 5 10 15

Ser Phe Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile

20 25 30

Lys Asp Ile

35

<210> 7

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 7

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Thr Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 8

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 8

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Gly Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 9

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 9

Met Thr Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 10

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 10

Met Asn Met Asn Asn Thr Lys Leu Asn Ala Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 11

<211> 31

<212> PRT

<213> A

<300>

<313> (1)..(31)

<400> 11

Met Asn Asn Thr Lys Leu Asn Ala Arg Ala Leu Pro Ser Phe Ile Asp

1 5 10 15

Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile

20 25 30

<210> 12

<211> 33

<212> PRT

<213> A

<400> 12

Met Asn Met Asn Asn Thr Lys Leu Ser Ala Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 13

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 13

Met Asn Met Asn Asn Thr Lys Leu Ser Ala Arg Ala Leu Pro Ser Leu

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 14

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 14

Met Asn Met Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 15

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 15

Met Asn Met Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 16

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 16

Met Asn Met Asn Asn Ala Lys Leu Asn Ala Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 17

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 17

Met Asn Met Asn Asn Thr Lys Leu Asn Ala Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Ile Gly Ile Lys Asp

20 25 30

Ile

<210> 18

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 18

Met Asn Met Asn Asn Ala Lys Leu Asn Ala Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Ile Gly Ile Lys Asp

20 25 30

Ile

<210> 19

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 19

Met Asn Lys Asn Asn Thr Lys Leu Asn Ala Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Ile Gly Ile Lys Asp

20 25 30

Ile

<210> 20

<211> 42

<212> PRT

<213> A

<300>

<313> (1)..(42)

<400> 20

Met Val Gln Lys Trp Met Gln Arg Met Ile Ile Val Asp Asn Asn Lys

1 5 10 15

Leu Asn Val Arg Ala Leu Pro Ser Phe Ile Asp Tyr Phe Asn Gly Ile

20 25 30

Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile

35 40

<210> 21

<211> 32

<212> PRT

<213> A

<300>

<313> (1)..(32)

<400> 21

Met Gln Lys Asn Asn Lys Leu Ser Val Lys Ala Leu Pro Ser Phe Ile

1 5 10 15

Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile

20 25 30

<210> 22

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 22

Met Asn Lys Asn Asn Thr Lys Leu Ser His Val Val Val Ile Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 23

<211> 34

<212> PRT

<213> A

<300>

<313> (1)..(34)

<400> 23

Met Asn Lys Asn Asn Thr Lys Leu Ser Asn Val Asn Glu Leu Ser Ser

1 5 10 15

Leu Ser Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys

20 25 30

Asp Ile

<210> 24

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 24

Met Asn Lys Asn Asn Thr Lys Leu Ser Lys Asn Glu Leu Ser Arg Leu

1 5 10 15

Ser Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 25

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 25

Met Asn Lys Asn Asn Thr Lys Leu Ser Lys Asn Glu Leu Ser Arg Leu

1 5 10 15

Ser Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 26

<211> 33

<212> PRT

<213> A

<300>

<313> (1)..(33)

<400> 26

Met Asn Lys Asn Asn Thr Lys Leu Ser Asn Phe Arg Cys Leu Tyr Leu

1 5 10 15

Val Glu Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile

<210> 27

<211> 11

<212> PRT

<213> A

<300>

<313> (1)..(11)

<400> 27

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg

1 5 10

<210> 28

<211> 10

<212> PRT

<213> A

<300>

<313> (1)..(10)

<400> 28

Met Ala Leu Pro Ser Phe Ile Asp Tyr Phe

1 5 10

<210> 29

<211> 14

<212> PRT

<213> A

<300>

<313> (1)..(14)

<400> 29

Met Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile

1 5 10

<210> 30

<211> 20

<212> PRT

<213> A

<300>

<313> (1)..(20)

<400> 30

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe

20

<210> 31

<211> 24

<212> PRT

<213> A

<300>

<313> (1)..(24)

<400> 31

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Asn Gly Ile Tyr Gly

1 5 10 15

Phe Ala Thr Gly Ile Lys Asp Ile

20

<210> 32

<211> 23

<212> PRT

<213> A

<300>

<313> (1)..(23)

<400> 32

Met Ala Leu Pro Ser Phe Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe

1 5 10 15

Ala Thr Gly Ile Lys Asp Ile

20

<210> 33

<211> 425

<212> PRT

<213> A

<300>

<313> (1)..(425)

<400> 33

Met Ser Thr Lys Trp Glu Lys Leu Glu Gly Asn Val Gly Val Leu Thr

1 5 10 15

Ile Glu Val Asp Ala Lys Glu Val Asn Asn Ser Ile Asp Ala Ala Phe

20 25 30

Lys Lys Val Val Lys Thr Ile Asn Val Pro Gly Phe Arg Lys Gly Lys

35 40 45

Met Pro Arg Pro Leu Phe Glu Gln Arg Phe Gly Ile Glu Ser Leu Tyr

50 55 60

Gln Asp Ala Leu Asp Ile Ile Leu Pro Lys Ala Tyr Gly Glu Ala Ile

65 70 75 80

Glu Glu Ala Gly Ile Phe Pro Val Asp His Pro Glu Ile Asp Ile Glu

85 90 95

Lys Phe Glu Lys Asn Ala Asn Leu Ile Phe Thr Ala Lys Val Thr Val

100 105 110

Lys Pro Glu Val Lys Leu Gly Glu Tyr Lys Gly Leu Ala Val Glu Lys

115 120 125

Val Glu Thr Thr Val Thr Asp Glu Asp Val Glu Asn Glu Leu Lys Ser

130 135 140

Leu Gln Glu Arg Gln Ala Glu Leu Val Val Lys Glu Glu Gly Thr Val

145 150 155 160

Glu Asn Gly Asp Thr Ala Val Ile Asp Phe Glu Gly Phe Val Asp Gly

165 170 175

Glu Ala Phe Glu Gly Gly Lys Gly Glu Asn Tyr Ser Leu Ala Ile Gly

180 185 190

Ser Gly Thr Phe Ile Pro Gly Phe Glu Glu Gln Val Ile Gly Leu Lys

195 200 205

Ser Gly Glu Ser Lys Asp Val Glu Val Ser Phe Pro Glu Glu Tyr His

210 215 220

Ala Ala Glu Leu Ala Gly Lys Pro Ala Thr Phe Lys Val Thr Ile His

225 230 235 240

Glu Ile Lys Thr Lys Glu Leu Pro Glu Leu Asn Asp Glu Phe Ala Lys

245 250 255

Glu Ala Asp Glu Ala Val Ala Thr Leu Asp Glu Leu Lys Ala Lys Leu

260 265 270

Arg Thr Asn Leu Glu Glu Gly Lys Lys His Glu Ala Glu His Lys Val

275 280 285

Arg Asp Glu Val Val Glu Leu Ala Ala Ala Asn Ala Glu Ile Glu Ile

290 295 300

Pro Glu Ala Met Ile Asn Thr Glu Leu Asp Arg Met Val Arg Glu Phe

305 310 315 320

Glu Gln Arg Leu Ser Gln Gln Gly Met Asn Leu Glu Leu Tyr Tyr Gln

325 330 335

Phe Thr Gly Thr Asp Ala Asp Lys Leu Lys Glu Gln Met Lys Glu Asp

340 345 350

Ala Gln Lys Arg Val Arg Ile Asn Leu Val Leu Glu Ala Ile Ile Glu

355 360 365

Ala Glu Asn Ile Glu Val Thr Glu Glu Glu Val Thr Ala Glu Val Glu

370 375 380

Lys Met Ala Glu Met Tyr Gly Met Pro Val Asp Ala Ile Lys Gln Ala

385 390 395 400

Leu Gly Ser Val Asp Ala Leu Ala Glu Asp Leu Lys Val Arg Lys Ala

405 410 415

Val Asp Phe Leu Val Glu Asn Ala Ala

420 425

<210> 34

<211> 155

<212> PRT

<213> A

<300>

<313> (1)..(155)

<400> 34

Met Ser Glu Gln Asn Asn Thr Glu Met Thr Phe Gln Ile Gln Arg Ile

1 5 10 15

Tyr Thr Lys Asp Ile Ser Phe Glu Ala Pro Asn Ala Pro His Val Phe

20 25 30

Gln Lys Asp Trp Gln Pro Glu Val Lys Leu Asp Leu Asp Thr Ala Ser

35 40 45

Ser Gln Leu Ala Asp Asp Val Tyr Glu Val Val Leu Arg Val Thr Val

50 55 60

Thr Ala Ser Leu Gly Glu Glu Thr Ala Phe Leu Cys Glu Val Gln Gln

65 70 75 80

Gly Gly Ile Phe Ser Ile Ala Gly Ile Glu Gly Thr Gln Met Ala His

85 90 95

Cys Leu Gly Ala Tyr Cys Pro Asn Ile Leu Phe Pro Tyr Ala Arg Glu

100 105 110

Cys Ile Thr Ser Met Val Ser Arg Gly Thr Phe Pro Gln Leu Asn Leu

115 120 125

Ala Pro Val Asn Phe Asp Ala Leu Phe Met Asn Tyr Leu Gln Gln Gln

130 135 140

Ala Gly Glu Gly Thr Glu Glu His Gln Asp Ala

145 150 155

<210> 35

<211> 155

<212> PRT

<213> A

<300>

<313> (1)..(155)

<400> 35

Met Ser Glu Gln Asn Asn Thr Glu Met Thr Phe Gln Ile Gln Arg Ile

1 5 10 15

Tyr Thr Lys Asp Ile Ser Phe Glu Ala Pro Asn Ala Pro His Val Phe

20 25 30

Gln Lys Asp Trp Gln Pro Glu Val Lys Leu Asp Leu Asp Thr Ala Ser

35 40 45

Ser Gln Leu Ala Asp Asp Val Tyr Glu Val Val Leu Arg Val Thr Val

50 55 60

Thr Ala Ser Leu Gly Glu Glu Thr Ala Phe Gln Cys Val Val Gln Gln

65 70 75 80

Gly Gly Ile Phe Ser Ile Ala Gly Ile Glu Gly Thr Gln Met Ala His

85 90 95

Cys Leu Gly Ala Tyr Cys Pro Asn Ile Leu Phe Pro Tyr Ala Arg Glu

100 105 110

Cys Ile Thr Ser Met Val Ser Arg Gly Thr Phe Pro Gln Leu Asn Leu

115 120 125

Ala Pro Val Asn Phe Asp Ala Leu Phe Met Asn Tyr Leu Gln Gln Gln

130 135 140

Ala Gly Glu Gly Thr Glu Glu His Gln Asp Ala

145 150 155

<210> 36

<211> 142

<212> PRT

<213> A

<300>

<313> (1)..(142)

<400> 36

Met Ser Glu Gln Asn Asn Thr Glu Met Thr Phe Gln Ile Gln Arg Ile

1 5 10 15

Tyr Thr Lys Asp Ile Ser Phe Glu Ala Pro Asn Ala Pro His Val Phe

20 25 30

Gln Lys Asp Trp Gln Pro Glu Val Lys Leu Asp Leu Asp Thr Ala Ser

35 40 45

Ser Gln Leu Ala Asp Asp Val Tyr Glu Val Val Leu Arg Val Thr Val

50 55 60

Thr Ala Ser Leu Gly Glu Glu Thr Ala Phe Leu Cys Glu Val Gln Gln

65 70 75 80

Gly Gly Ile Phe Ser Ile Ala Gly Ile Glu Gly Thr Gln Met Ala His

85 90 95

Cys Leu Gly Ala Tyr Cys Pro Asn Ile Leu Phe Pro Tyr Ala Arg Glu

100 105 110

Cys Ile Thr Ser Met Val Ser Arg Gly Thr Phe Pro Gln Leu Asn Leu

115 120 125

Ala Pro Val Asn Phe Asp Ala Leu Phe Met Asn Tyr Leu Gln

130 135 140

<210> 37

<211> 142

<212> PRT

<213> A

<300>

<313> (1)..(142)

<400> 37

Met Ser Glu Gln Asn Asn Thr Glu Met Thr Phe Gln Ile Gln Arg Ile

1 5 10 15

Tyr Thr Lys Asp Ile Ser Phe Glu Ala Pro Asn Ala Pro His Val Phe

20 25 30

Gln Lys Asp Trp Gln Pro Glu Val Lys Leu Asp Leu Asp Thr Ala Ser

35 40 45

Ser Gln Leu Ala Asp Asp Val Tyr Glu Val Val Leu Arg Val Thr Val

50 55 60

Thr Ala Ser Leu Gly Glu Glu Thr Ala Phe Gln Cys Val Val Gln Gln

65 70 75 80

Gly Gly Ile Phe Ser Ile Ala Gly Ile Glu Gly Thr Gln Met Ala His

85 90 95

Cys Leu Gly Ala Tyr Cys Pro Asn Ile Leu Phe Pro Tyr Ala Arg Glu

100 105 110

Cys Ile Thr Ser Met Val Ser Arg Gly Thr Phe Pro Gln Leu Asn Leu

115 120 125

Ala Pro Val Asn Phe Asp Ala Leu Phe Met Asn Tyr Leu Gln

130 135 140

<210> 38

<211> 835

<212> PRT

<213> A

<300>

<313> (1)..(835)

<400> 38

Met Ile Gly Ile Leu Lys Lys Val Phe Asp Val Asn Gln Arg Gln Ile

1 5 10 15

Lys Arg Met Gln Lys Thr Val Glu Gln Ile Asp Ala Leu Glu Ser Ser

20 25 30

Ile Lys Pro Leu Thr Asp Glu Gln Leu Lys Gly Lys Thr Leu Glu Phe

35 40 45

Lys Glu Arg Leu Thr Lys Gly Glu Thr Val Asp Asp Leu Leu Pro Glu

50 55 60

Ala Phe Ala Val Val Arg Glu Ala Ala Thr Arg Val Leu Gly Met Arg

65 70 75 80

Pro Tyr Gly Val Gln Leu Met Gly Gly Ile Ala Leu His Glu Gly Asn

85 90 95

Ile Ser Glu Met Lys Thr Gly Glu Gly Lys Thr Leu Thr Ser Thr Leu

100 105 110

Pro Val Tyr Leu Asn Ala Leu Thr Gly Lys Gly Val His Val Val Thr

115 120 125

Val Asn Glu Tyr Leu Ala Gln Arg Asp Ala Ser Glu Met Gly Gln Leu

130 135 140

His Glu Phe Leu Gly Leu Thr Val Gly Ile Asn Leu Asn Ser Met Ser

145 150 155 160

Arg Glu Glu Lys Gln Glu Ala Tyr Ala Ala Asp Ile Thr Tyr Ser Thr

165 170 175

Asn Asn Glu Leu Gly Phe Asp Tyr Leu Arg Asp Asn Met Val Leu Tyr

180 185 190

Lys Glu Gln Cys Val Gln Arg Pro Leu His Phe Ala Ile Ile Asp Glu

195 200 205

Val Asp Ser Ile Leu Val Asp Glu Ala Arg Thr Pro Leu Ile Ile Ser

210 215 220

Gly Gln Ala Gln Lys Ser Thr Glu Leu Tyr Met Phe Ala Asn Ala Phe

225 230 235 240

Val Arg Thr Leu Glu Asn Glu Lys Asp Tyr Ser Phe Asp Val Lys Thr

245 250 255

Lys Asn Val Met Leu Thr Glu Asp Gly Ile Thr Lys Ala Glu Lys Ala

260 265 270

Phe His Ile Glu Asn Leu Phe Asp Leu Lys His Val Ala Leu Leu His

275 280 285

His Ile Asn Gln Gly Leu Arg Ala His Val Val Met His Arg Asp Thr

290 295 300

Asp Tyr Val Val Gln Glu Gly Glu Ile Val Ile Val Asp Gln Phe Thr

305 310 315 320

Gly Arg Leu Met Lys Gly Arg Arg Tyr Ser Glu Gly Leu His Gln Ala

325 330 335

Ile Glu Ala Lys Glu Gly Val Glu Ile Gln Asn Glu Ser Met Thr Leu

340 345 350

Ala Thr Ile Thr Phe Gln Asn Tyr Phe Arg Met Tyr Glu Lys Leu Ser

355 360 365

Gly Met Thr Gly Thr Ala Lys Thr Glu Glu Glu Glu Phe Arg Asn Ile

370 375 380

Tyr Asn Met Asn Val Ile Val Ile Pro Thr Asn Lys Pro Ile Ile Arg

385 390 395 400

Asp Asp Arg Ala Asp Leu Ile Phe Lys Ser Met Glu Gly Lys Phe Asn

405 410 415

Ala Val Val Glu Asp Ile Val Asn Arg His Lys Lys Gly Gln Pro Val

420 425 430

Leu Val Gly Thr Val Ala Ile Glu Thr Ser Glu Leu Ile Ser Lys Met

435 440 445

Leu Thr Arg Lys Gly Val Arg His Asn Ile Leu Asn Ala Lys Asn His

450 455 460

Ala Arg Glu Ala Asp Ile Ile Ala Glu Ala Gly Ile Lys Gly Ala Val

465 470 475 480

Thr Ile Ala Thr Asn Met Ala Gly Arg Gly Thr Asp Ile Lys Leu Gly

485 490 495

Asp Asp Val Lys Asn Val Gly Leu Ala Val Ile Gly Thr Glu Arg His

500 505 510

Glu Ser Arg Arg Ile Asp Asn Gln Leu Arg Gly Arg Ala Gly Arg Gln

515 520 525

Gly Asp Pro Gly Val Thr Gln Phe Tyr Leu Ser Met Glu Asp Glu Leu

530 535 540

Met Arg Arg Phe Gly Ser Asp Asn Met Lys Ala Met Met Asp Arg Leu

545 550 555 560

Gly Met Asp Asp Ser Gln Pro Ile Glu Ser Lys Met Val Ser Arg Ala

565 570 575

Val Glu Ser Ala Gln Lys Arg Val Glu Gly Asn Asn Tyr Asp Ala Arg

580 585 590

Lys Gln Leu Leu Gln Tyr Asp Asp Val Leu Arg Gln Gln Arg Glu Val

595 600 605

Ile Tyr Lys Gln Arg Gln Glu Val Met Glu Ser Asp Asn Leu Arg Gly

610 615 620

Ile Ile Glu Gly Met Met Lys Ser Thr Val Glu Arg Ala Val Ala Leu

625 630 635 640

His Thr Gln Glu Glu Ile Glu Glu Asp Trp Asn Ile Lys Gly Leu Val

645 650 655

Asp Tyr Leu Asn Thr Asn Leu Leu Gln Asp Gly Asp Val Lys Glu Glu

660 665 670

Glu Leu Arg Arg Leu Ala Pro Glu Glu Met Ser Glu Pro Ile Ile Ala

675 680 685

Lys Leu Ile Glu Arg Tyr Asn Glu Lys Glu Lys Leu Met Pro Glu Glu

690 695 700

Gln Met Arg Glu Phe Glu Lys Val Val Val Phe Arg Val Val Asp Thr

705 710 715 720

Lys Trp Thr Glu His Ile Asp Ala Met Asp His Leu Arg Glu Gly Ile

725 730 735

His Leu Arg Ala Tyr Gly Gln Ile Asp Pro Leu Arg Glu Tyr Gln Met

740 745 750

Glu Gly Phe Ala Met Phe Glu Ser Met Val Ala Ser Ile Glu Glu Glu

755 760 765

Ile Ser Arg Tyr Ile Met Lys Ala Glu Ile Glu Gln Asn Leu Glu Arg

770 775 780

Gln Glu Val Val Gln Gly Glu Ala Val His Pro Ser Ser Asp Gly Glu

785 790 795 800

Glu Ala Lys Lys Lys Pro Val Val Lys Gly Asp Gln Val Gly Arg Asn

805 810 815

Asp Leu Cys Lys Cys Gly Ser Gly Lys Lys Tyr Lys Asn Cys Cys Gly

820 825 830

Ile Val Gln

835

Claims

1. The method for simultaneously enhancing the expression quantity and the solubility of the target protein in a prokaryotic system is characterized in that a specific guide sequence is used as a novel fusion tag to form a fusion gene with a polynucleotide sequence for encoding the target protein, and the fusion gene and a chaperone Trigger gene are co-expressed in the same prokaryotic expression system, so that the expression quantity and the solubility of the target protein can be enhanced;

the amino acid sequence of the specific guide sequence is selected from the group consisting of: any amino acid sequence shown in SEQ ID NO. 1-32;

the polypeptide sequence of the molecular chaperone Trigger gene is shown as SEQ ID NO. 33.

2. The method for simultaneously enhancing the expression level and the solubility of a protein of interest in a prokaryotic system according to claim 1, wherein the specific guide sequence is located at the N-terminal of the protein of interest, and the specific guide sequence can be directly connected, or a spacer sequence can be present between the specific guide sequence and the specific guide sequence.

3. The method for simultaneously enhancing the expression level and the solubility of a protein of interest in a prokaryotic system according to claim 2, wherein the spacer sequence is an amino acid sequence encoded by a polynucleotide sequence of a recognition site for a restriction enzyme.

4. The method for simultaneously enhancing the expression level and the solubility of a target protein in a prokaryotic system according to claim 1, wherein the polynucleotide sequence of the specific guide sequence can interact with a chaperone Trigger gene.

5. The method for simultaneously enhancing the expression level and the solubility of a protein of interest in a prokaryotic system according to claim 1, wherein the polynucleotide sequence encoding the chaperone Trigger gene and the fusion gene may be located in the same DNA molecule or DNA vector or may be located in different DNA molecules or DNA vectors, respectively, but are not translated independently in one reading frame.