Escherichia coli visual expression system for promoting soluble expression of recombinant proteins and construction method and application thereof

Abstract

The application discloses an E. coli visual expression system for promoting soluble expression of recombinant proteins and a construction method and application thereof. The application realizes fusion expression of target proteins by constructing a recombinant expression vector containing an enhanced green fluorescent protein (eGFP) tag and a ubiquitin-like modifier (SUMO) tag. The system constructed by the application organically combines visual monitoring of protein expression with high solubility expression, effectively solves the problems of complex protein expression difficulty and low folding efficiency, and has important application value in the fields of biological medicine development, proteomics research and industrial enzyme preparation production.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and recombinant protein technology, and specifically relates to an Escherichia coli visualization expression system for promoting soluble expression of recombinant proteins, its construction method and application. Background Technology

[0002] Recombinant protein technology is a core tool in modern biotechnology and biomedicine, widely used in biopharmaceutical development, diagnostic reagent production, and basic scientific research. The *Escherichia coli* expression system, due to its clear genetic background, ease of operation, rapid growth, and low cost, has become the most widely used prokaryotic expression host. However, the *E. coli* expression system often faces a common technical challenge when processing exogenous proteins, especially eukaryotic proteins—the target protein aggregates in large quantities as insoluble inclusion bodies, losing its native conformation and biological activity. Numerous studies have shown that the formation of inclusion bodies in *E. coli* is mainly due to the folding rate of nascent peptide chains exceeding their correct folding capacity, leading to the aggregation of misfolded intermediates into insoluble particles. This problem severely restricts the soluble yield and subsequent applications of recombinant proteins, and remains a long-standing technical bottleneck in the field of recombinant protein expression.

[0003] Small ubiquitin-like modifier (SUMO) tags are a widely used type of fusion tag for promoting solubility. SUMO tags originate from SUMO proteins, which are abundant in eukaryotes and whose natural function is involved in post-translational modification of proteins. In recombinant protein research, SUMO tags are typically attached to the N-terminus of the target protein in the form of a fusion protein. The core technological advantages of SUMO tags include: firstly, their highly conserved three-dimensional folded structure provides a favorable spatial environment for downstream target proteins during translation and folding, helping them maintain a soluble state; secondly, SUMO tags can act as molecular chaperones, reducing protein aggregation and significantly improving the solubility and yield of the target protein; and thirdly, SUMO tags can be specifically recognized and precisely cleaved by SUMO proteases (such as Ulp1), which recognize the three-dimensional structure of SUMO rather than its sequence motif, thus forming the actual N-terminus of the target protein after processing. Studies have also shown that SUMO tags have good tolerance to high temperatures and proteases, and their effect on enhancing protein solubility in recombinant protein production is significantly better than that of His tags.

[0004] Enhanced Green Fluorescent Protein (eGFP) is a fluorescent protein mutant derived from jellyfish, exhibiting excellent autofluorescence properties in both prokaryotic and eukaryotic expression systems. Studies have shown that the correct folding and fluorescence of the GFP fused to the C-terminus of the target protein depend on proper folding; therefore, only folded fusion proteins will fluoresce. Conversely, if the fusion protein is present in inclusion bodies, GFP cannot fold correctly and will not fluoresce. Therefore, fusing eGFP with the target protein not only allows for direct monitoring of protein expression levels through fluorescence intensity but also leverages its high solubility to help maintain the target protein in a soluble state. Research shows that eGFP as a fusion tag can significantly improve the expression level of the fusion protein (by approximately 53%), solubility (by approximately 77%), and stability. Linking eGFP to the target protein via a flexible linker allows for visual tracking of the protein without interfering with its function. However, the large molecular weight of the eGFP tag (approximately 27 kDa) may negatively impact downstream applications of the target protein in the long term, and eGFP alone offers limited assistance in ensuring correct folding of the target protein. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a visual expression system for Escherichia coli that promotes the soluble expression of recombinant proteins.

[0006] Another object of the present invention is to provide the application of the Escherichia coli visual expression system that promotes the soluble expression of recombinant proteins.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A visual expression system for promoting the soluble expression of recombinant proteins in *E. coli* is constructed using any of the following methods:

[0009] Method 1:

[0010] (1) Linearize the recombinant expression vector containing green fluorescent protein (eGFP) and the soluble tag SUMO to obtain a linearized recombinant expression vector;

[0011] (2) The target gene fragment is ligated into a linearized recombinant expression vector to obtain the Escherichia coli visual expression system that promotes the soluble expression of recombinant proteins;

[0012] Method 2:

[0013] (3) ① Connect the gene sequence of green fluorescent protein (eGFP), the gene sequence of the soluble tag SUMO and the target gene sequence in sequence to obtain target fragment 1 (the target gene fragment containing eGFP and SUMO).

[0014] Alternatively: ② Connect the gene sequence of green fluorescent protein (eGFP), the gene sequence of the soluble tag SUMO, the His tag sequence and the target gene sequence in sequence to obtain target fragment 2 (the target gene fragment containing eGFP, SUMO and His tags).

[0015] (4) Connect the target fragment 1 or the target fragment 2 to a linearized prokaryotic expression vector to obtain the Escherichia coli visual expression system that promotes the soluble expression of recombinant proteins.

[0016] The amino acid sequence of the green fluorescent protein (eGFP) described in step (1) is shown in SEQ ID NO.1.

[0017] The amino acid sequence of the dissolving tag SUMO described in step (1) is shown in SEQ ID NO.2.

[0018] The amino acid sequences of the green fluorescent protein (eGFP) and the dissolution tag SUMO (eGFP-SUMO) mentioned in step (1) are shown as positions 1-339 in SEQ ID NO.3, and their nucleotide sequences are shown as positions 1-1017 bp in SEQ ID NO.4.

[0019] The starting vector for the recombinant expression vector mentioned in step (1) is the pET-21a series vector; preferably the pET-21a(+) vector.

[0020] The recombinant expression vector containing green fluorescent protein (eGFP) and the dissolution tag SUMO mentioned in step (1) is preferably pET-21a(+)-eGFP-SUMO.

[0021] In step (1), the linearization process can be obtained by designing corresponding primer sequences according to the recombinant expression vector and amplifying it linearly; for example, for the recombinant expression vector pET-21a(+)-eGFP-SUMO, primer pairs as shown in SEQ ID NO.11 and SEQ ID NO.12 can be designed to amplify the linearized pET-21a(+)-eGFP-SUMO vector; or primer pairs as shown in SEQ ID NO.19 and SEQ ID NO.20, and SEQ ID NO.21 and SEQ ID NO.22 can be designed to amplify the linearized vector fragments 1 and 2.

[0022] The protein encoded by the target gene in steps (2) and (3) is at least one of FGF21, HSA-Nb80, SAF2 and SAF2 mutant; preferably at least one of FGF21, HSA-Nb80 and SAF2 mutant.

[0023] The amino acid sequence of FGF21 is shown in positions 351-545 of SEQ ID NO.3, and the nucleotide sequence is shown in positions 1053-1635 of SEQ ID NO.4.

[0024] The amino acid sequence of HSA-Nb80 is shown in positions 351-486 of SEQ ID NO.5, and the nucleotide sequence is shown in positions 1053-1458 bp of SEQ ID NO.6.

[0025] The amino acid sequence of the SAF2 is shown in positions 341-576 of SEQ ID NO.7, and the nucleotide sequence is shown in positions 1021-1728 of SEQ ID NO.8.

[0026] The SAF2 mutant is characterized by the mutation of two cysteine ​​residues (C) at positions 121 and 139 of SAF2 to serine residues (S). Its amino acid sequence is shown as positions 341-576 in SEQ ID NO.9, and its nucleotide sequence is shown as positions 1021-1728 bp in SEQ ID NO.10.

[0027] The target gene fragment mentioned in step (2) is preferably a target gene fragment with a homologous arm of a prokaryotic expression vector; more preferably, it is at least one of the following: FGF21 fragment, HSA-Nb80 fragment, SAF2 fragment, and SAF2 mutant fragment; wherein,

[0028] The FGF21 fragment was amplified using pUC-GW-Kan-FGF21 plasmid as a template and primers SEQ ID NO.13 and SEQ ID NO.14.

[0029] The HSA-Nb80 fragment was amplified using pET-21a(+)-HSA-Nb80 plasmid as a template and primers SEQ ID NO.15 and SEQ ID NO.16.

[0030] The SAF2 fragment was amplified using pET-21a(+)-SAF2 plasmid as a template and primers SEQ ID NO.17 and SEQ ID NO.18.

[0031] The SAF2 mutant fragment was amplified using the pET-21a(+)-SAF2 mutant plasmid as a template and primers SEQ ID NO.23 and SEQ ID NO.24.

[0032] In step (3), the gene sequence of green fluorescent protein (eGFP), the gene sequence of the dissolution tag SUMO, and the target gene sequence can be directly linked in sequence. To facilitate the subsequent purification of recombinant protein, a His tag can also be added between the gene sequence of the dissolution tag SUMO and the target gene sequence, wherein the amino acid sequence of the His tag is as follows: KSSHHHHHHGS.

[0033] The target fragment 1 (containing the target gene fragment of eGFP and SUMO) mentioned in step (3) is preferably eGFP-SUMO-SAF2 or an eGFP-SUMO-SAF2 mutant; wherein,

[0034] The protein sequence encoded by the eGFP-SUMO-SAF2 is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8;

[0035] The protein sequence encoded by the eGFP-SUMO-SAF2 mutant is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10.

[0036] The target fragment 2 (the target gene fragment containing eGFP, SUMO, and His tags) mentioned in step (3) is preferably eGFP-SUMO-FGF21 or eGFP-SUMO-HSA-Nb80; wherein,

[0037] The protein sequence encoded by the eGFP-SUMO-FGF21 is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4;

[0038] The protein sequence encoded by the eGFP-SUMO-HSA-Nb80 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0039] The prokaryotic expression vector mentioned in step (4) is a pET-21a series vector; preferably a pET-21a(+) vector.

[0040] Application of the Escherichia coli visualization expression system for promoting soluble expression of recombinant proteins in soluble expression of recombinant proteins.

[0041] The recombinant protein includes at least one of FGF21, HSA-Nb80, SAF2, or a SAF2 mutant.

[0042] The SAF2 mutant is characterized by the mutation of two cysteine ​​residues (C) at positions 121 and 139 of SAF2 to serine residues (S).

[0043] The present invention has the following advantages and effects compared with the prior art:

[0044] 1. This invention provides an *E. coli* protein fusion expression system based on an eGFP-SUMO dual-tag. This system achieves the fusion expression of the target protein by constructing a recombinant expression vector containing both an enhanced green fluorescent protein (eGFP) tag and a ubiquitin-associated modifier (SUMO) tag. The introduction of the eGFP tag enables real-time, in-situ monitoring of the target protein's expression level, spatiotemporal distribution, and enrichment during purification by fluorescence signal intensity. The SUMO tag, as a highly efficient molecular chaperone, significantly improves the solubility of heterologous proteins in prokaryotic or eukaryotic systems, promotes correct folding of the target protein, and prevents inclusion body formation. This invention organically combines visualized monitoring of protein expression with highly soluble expression, effectively solving the problems of difficult expression and low folding efficiency of complex proteins. It has significant application value in biopharmaceutical development, proteomics research, and industrial enzyme production.

[0045] 2. This invention uses the pET-21a(+) vector as an example, introducing eGFP (green fluorescent protein) and the soluble tag SUMO to construct the pET-21a(+)-eGFP-SUMO expression vector. Expression analysis revealed that it exhibited good expression levels and soluble expression. Therefore, eGFP-SUMO (GS) serves as a molecular chaperone for co-expressing the target protein, resulting in highly visualized and soluble expression of the target protein.

[0046] 3. The fluorescent expression system constructed in this invention fills the gap in the field where visible tags or tracking systems cannot be conveniently used to promote the amplification and purification of colorless molecules, and is beneficial for monitoring the production of soluble proteins using E. coli systems. Attached Figure Description

[0047] Figure 1 This is a diagram of the PCR amplification results of the target gene products pET-21a(+)-eGFP-SUMO and FGF21 (M: DNA marker; Lane 1: pET-21a(+)-eGFP-SUMO linear vector; Lane 2: FGF21 gene fragment containing the homologous arm of the pET-21a(+)-eGFP-SUMO linear vector).

[0048] Figure 2 This is a map of the pET-21a(+)-eGFP-SUMO-FGF21 recombinant plasmid and the results of colony PCR identification; among them,

[0049] (A) is the pET-21a(+)-eGFP-SUMO-FGF21 plasmid map; (B) is the pET-21a(+)-eGFP-SUMO-FGF21 positive clone band (n=5).

[0050] Figure 3 This is a graph showing the expression results of pET-21a(+)-eGFP-SUMO-FGF21 analyzed by SDS-PAGE (M: protein marker; lane 1: bacterial sample before induction; lanes 2-5: bacterial sample after induction).

[0051] Figure 4 This is an SDS-PAGE analysis of the solubility and enzyme digestion efficiency of recombinant protein eGFP-SUMO-FGF21 (M: protein marker; lane 1: pre-induction strain sample; lane 2: post-induction strain sample; lane 3: post-induction strain lysate sample; lane 4: post-induction strain lysate supernatant; lane 5: post-induction strain lysate precipitate; lane 6: post-induction strain enzyme digestion lysate sample; lane 7: post-induction strain enzyme digestion lysate supernatant; lane 8: post-induction strain enzyme digestion lysate precipitate).

[0052] Figure 5 This is a graph showing the purification results of recombinant protein FGF21 (M: protein marker; lane 1: supernatant after induction and enzymatic digestion; lane 2: DEAE column elution peak; lanes 3-4: Ni column elution peak; lanes 5-6: Ni column elution peak; lanes 7-9: Ni column elution peak; lanes 10-11: Phenyl column elution peak).

[0053] Figure 6 This is the SDS-PAGE identification result of the recombinant protein (M: protein marker; lane 1: recombinant protein FGF21).

[0054] Figure 7 This is a Western blot diagram of the recombinant protein (M: protein marker; lane 1: recombinant protein FGF21).

[0055] Figure 8 This is a diagram of the PCR amplification results of the target gene products pET-21a(+)-eGFP-SUMO and HSA-Nb80 (M: DNA marker; Lane 1: pET-21a(+)-eGFP-SUMO linear vector; Lane 2: HSA-Nb80 gene fragment containing homologous arms of the pET-21a(+)-eGFP-SUMO linear vector).

[0056] Figure 9The images show the patterns and colony PCR identification results of the recombinant plasmids pET-21a(+)-eGFP-SUMO-FGF21 and pET-21a(+)-eGFP-SUMO-HSA-Nb80; where (A) is the pattern of the pET-21a(+)-eGFP-SUMO-HSA-Nb80 plasmid; and (B) is the positive clone band of pET-21a(+)-eGFP-SUMO-FGF21.

[0057] Figure 10 This is a graph showing the expression results of pET-21a(+)-eGFP-SUMO-HSA-Nb80 analyzed by SDS-PAGE (M: protein marker; lane 1: bacterial sample before induction; lanes 2-5: bacterial sample after induction).

[0058] Figure 11 This is an SDS-PAGE analysis of the solubility and enzyme digestion efficiency of recombinant protein eGFP-SUMO-HSA-Nb80 (M: protein marker; lane 1: pre-induction strain sample; lane 2: post-induction strain sample; lane 3: post-induction strain lysate sample; lane 4: post-induction strain lysate supernatant; lane 5: post-induction strain lysate precipitate; lane 6: post-induction strain enzyme digestion lysate sample; lane 7: post-induction strain enzyme digestion lysate supernatant; lane 8: post-induction strain enzyme digestion lysate precipitate).

[0059] Figure 12 This is a graph showing the purification results of recombinant protein HSA-Nb80 (M: protein marker; lane 1: supernatant after induction and enzymatic digestion; lane 2: DEAE column crossover peak; lanes 3-4: Ni column crossover peak; lanes 5-7: Ni column elution peak; lanes 8-10: Ni column elution peak).

[0060] Figure 13 This is an image showing the SDS-PAGE identification results of the recombinant protein (M: protein marker; lane 1: recombinant protein HSA-Nb80).

[0061] Figure 14 This is a Western blot diagram of the recombinant protein (M: protein marker; lane 1: recombinant protein HSA-Nb80).

[0062] Figure 15 This is an electrophoresis image of the PCR amplification of the eGFP-SUMO-SAF2 fusion gene (M: DNA marker; lane 1: target gene SAF2; lanes 2-3: linearized vector fragments 1 and 2).

[0063] Figure 16This is a diagram showing the colony PCR identification results of the pET-21a(+)-eGFP-SUMO-SAF2 recombinant plasmid.

[0064] Figure 17 This is a graph showing the expression analysis results of the pET-21a(+)-eGFP-SUMO-SAF2 fusion protein (in the graph, (-): before induction; T: whole cell disruption; S: supernatant after disruption; P: precipitate after disruption).

[0065] Figure 18 This is an electrophoresis image of the PCR amplification of the eGFP-SUMO-SAF2 mutant fusion gene (M: DNA marker; lane 1: SAF2 mutant; lanes 2-3: linearized vector fragments 1 and 2).

[0066] Figure 19 This is a diagram showing the colony PCR identification results of the pET-21a(+)-eGFP-SUMO-SAF2 mutant recombinant plasmid.

[0067] Figure 20 The image shows the expression analysis results of the eGFP-SUMO-SAF2 mutant. In the image, A is the expression bacterial culture (1: whole bacterial culture before lysis; 2: whole bacterial culture after lysis; 3: supernatant after lysis; 4: precipitate after lysis); B is the electrophoresis image of the GFP-SUMO-SAF2 mutant expression analysis ((-): before induction; T: whole bacterial culture lysis; S: supernatant after lysis; P: precipitate after lysis).

[0068] Figure 21 This is a CM ion exchange chromatography electrophoresis image of the soluble SAF2 mutant (M: protein marker; lane 1: dissolved sample; lane 2: sample loading and exit; lane 3: washing; lane 4: 0.1 mol / L NaCl; lane 5: 0.15 mol / L NaCl; lane 6: 0.2 mol / L NaCl; lane 7: 0.3 mol / L NaCl; lane 8: 0.4 mol / L NaCl; lane 9: 0.5 mol / L NaCl; lane 10: 0.7 mol / L NaCl; lane 11: 0.5 mol / L NaOH).

[0069] Figure 22This is an HP affinity electrophoresis image of the soluble SAF2 mutant (M: protein marker; lane 1: dissolved sample; lane 2: sample loading and exit; lane 3: washing; lane 4: 0.2 mol / L NaCl; lane 5: 0.3 mol / L NaCl; lane 6: 0.4 mol / L NaCl; lane 7: 0.5 mol / L NaCl; lane 8: 0.7 mol / L NaCl; lane 9: 1 mol / L NaCl; lane 10: 0.1 mol / L NaOH).

[0070] Figure 23 This is the pET-21a(+)-eGFP-SUMO plasmid map. Detailed Implementation

[0071] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0072] Example 1: Expression and purification of pET-21a(+)-eGFP-SUMO-FGF21

[0073] 1.1 Experimental Materials

[0074] Experimental materials: pET-21a(+)-eGFP-SUMO (synthesized by a commissioned biotechnology company; the eGFP-SUMO sequence was synthesized by Nanjing GenScript Biotech Co., Ltd. and cloned into the vector pET-21a(+)) (plasmid map as shown) Figure 23The following were used: pUC-GW-Kan-FGF21 plasmid (synthesized by a commissioned biotechnology company: the FGF21 sequence was synthesized by Suzhou Genewise Biotechnology Co., Ltd. and cloned into the vector pUC-GW-Kan); E. coli DH5α competent cells (purchased from TransGen Biotech Co., Ltd.); E. coli BL21(DE3) competent cells (purchased from TransGen Biotech Co., Ltd.); pET-21a(+)-SUMOE plasmid (SUMO protease expression plasmid pET-21a(+)-SUMOE synthesized by Nanjing Genscript Biotech Co., Ltd.); ampicillin (Sangon Biotech Co., Ltd.); isopropyl-β-D-thiogalactoside (Guangzhou Jiebes Biotechnology Co., Ltd.); HiTrap DEAE SepharoseFF (purchased from Cityva); HiTrap Phenyl FF (High Sub) (purchased from Cityva); Ni-NTA Agarose Resin (purchased from Shanghai Yisheng Biotechnology Co., Ltd.); and FGF21 monoclonal antibody (purchased from ABclonal Biotechnology Co., Ltd.).

[0075] 1.2 PCR amplification of genes

[0076] Experimental Methods: To obtain the linearized vector of pET-21a(+)-eGFP-SUMO and the target gene fragment of FGF21 (fibroblast growth factor 21) with the corresponding homologous arm, we used pET-21a(+)-eGFP-SUMO plasmid and pUC-GW-Kan-FGF21 plasmid as templates, respectively, and performed PCR amplification of pET-21a(+)-eGFP-SUMO and FGF21 using specific primers. The amplification products were analyzed by 1.5% agarose gel electrophoresis. The primer sequences involved are shown in Table 1.

[0077] Table 1

[0078]

[0079] eGFP amino acid sequence (SEQ ID NO.1):

[0080] VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGIE;

[0081] SUMO amino acid sequence (SEQ ID NO.2):

[0082] MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG;

[0083] eGFP-SUMO-FGF21 amino acid sequence (amino acids 1-339 of the sequence are the eGFP-SUMO sequence, amino acids 340-350 (KSSHHHHHHGS) are the His tag sequence, and amino acids 351-545 are the FGF21 sequence) (SEQ ID NO. 3):

[0084] VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGIEMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGKSSHHHHHHGSMSGSETPGTSESATPESMDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGGSQGRSPSYES;

[0085] eGFP-SUMO-FGF21 nucleotide sequence (the 1st to 1017th bp in the sequence is the eGFP-SUMO gene sequence, and the 1053rd to 1635th bp is the FGF21 gene sequence) (SEQ ID NO.4):

[0086]

[0087] Result: As Figure 1 The results showed that lane 1 contained the pET-21a(+)-eGFP-SUMO linear vector, and lane 2 contained the FGF21 gene fragment containing the homologous arm of the pET-21a(+)-eGFP-SUMO linear vector, located at 6383 bp and 618 bp, respectively, which were consistent with the expected fragment sizes.

[0088] 1.3 Validation of positive clones:

[0089] Methods: Homologous recombination technology was used to ligate the amplified pET-21a(+)-eGFP-SUMO vector fragment with the FGF21 target gene fragment (containing the homologous arm of the pET-21a(+)-eGFP-SUMO linear vector) to obtain the pET-21a(+)-eGFP-SUMO-FGF21 recombinant plasmid (plasmid map shown in Figure 1). Figure 2 (As shown in (A)), then transformed into Escherichia coli DH5α competent cells, and then single colonies were picked from solid LB plates containing ampicillin (100 μg / mL) resistance, and colony PCR was performed using specific primers for amplifying FGF21 (Table 1).

[0090] Results: The PCR products were analyzed by 1.5% agarose gel electrophoresis, and the results are as follows. Figure 2 As shown in (B), all selected monoclonal antibodies contained positive recombinants, and the amplified bands were consistent with the expected size.

[0091] 1.4 Expression analysis of the fusion protein eGFP-SUMO-FGF21

[0092] Methods: *E. coli* BL21(DE3) containing the correctly sequenced pET-21a(+)-eGFP-SUMO-FGF21 recombinant plasmid were inoculated into LB broth (containing 100 μg / mL ampicillin) and cultured overnight at 37 ℃ and 220 rpm. The next day, the overnight culture was transferred to fresh LB broth (containing 100 μg / mL ampicillin) at a ratio of 1:100 (volume / volume). When the culture reached the logarithmic growth phase, a small amount of the culture was collected as a pre-induction sample. IPTG (isopropyl-β-D-thiogalactoside) was added to the remaining culture to induce expression. After induction at 37 ℃ for 4 h, the bacterial culture was collected by centrifugation as a post-induction sample. The pre-induction sample was used as a control, and SDS-PAGE electrophoresis was performed for detection.

[0093] Results: 12% SDS-PAGE electrophoresis showed that the recombinant protein eGFP-SUMO-FGF21 had a distinct band around 60 kDa in lanes 2-5. Figure 3 And all of them are consistent with the expected relative molecular weight.

[0094] 1.5 Solubility and Enzyme Digestion Analysis of Recombinant Protein eGFP-SUMO-FGF21

[0095] Methods: Induced *E. coli* BL21(DE3) containing the recombinant plasmid pET-21a(+)-eGFP-SUMO-FGF21 (named *E. coli* BL21(DE3) / pET-21a(+)-eGFP-SUMO-FGF21) were resuspended, homogenized using a high-pressure homogenizer, and centrifuged at 12,000 rpm at 4 °C. Pre-induction bacterial samples, post-induction bacterial samples, post-induction bacterial lysates, and the supernatant and precipitate after centrifugation of post-induction bacterial lysates were analyzed by SDS-PAGE electrophoresis to determine the major expression form of the target protein before enzyme digestion. Meanwhile, another portion of induced E. coli BL21(DE3) / pET-21a(+)-eGFP-SUMO-FGF21 cells and induced E. coli BL21(DE3) / pET-21a(+)-SUMOE cells (the pET-21a(+)-SUMOE plasmid was transformed into E. coli BL21(DE3), and the induction conditions were the same as described in 1.4 above) were mixed at a ratio of 10:1 by wet weight of bacterial sludge, resuspended, and then homogenized using a high-pressure homogenizer. After high-speed centrifugation at 4 ℃ and 12000 rpm, the enzyme digestion lysate, the supernatant of the lysate after centrifugation, and the precipitate were taken for SDS-PAGE electrophoresis analysis to determine the enzyme digestion effect and the main expression form of the target protein after enzyme digestion.

[0096] Results: SDS-PAGE electrophoresis results showed that... Figure 4 In lanes 2 and 3, distinct protein expression bands were observed around 60 kDa in both the induced E. coli BL21(DE3) / pET-21a(+)-eGFP-SUMO-FGF21 bacterial cell and cell lysate samples, consistent with the expected molecular weight of the fusion protein eGFP-SUMO-FGF21. Meanwhile, in... Figure 4 In lane 4, a clear protein expression bar was observed in the supernatant sample of the bacterial lysate at approximately 60 kDa. Figure 4 In lane 5, the lysate precipitate of the above-mentioned bacterial cells showed almost no obvious protein expression bands around 60 kDa. The results indicate that the recombinant protein eGFP-SUMO-FGF21, before enzyme digestion, was mostly present in a soluble form.

[0097] The results after enzyme digestion showed that, Figure 4 In lane 6 of the swim grout, two distinct bands were observed in the lysate sample of E. coli BL21(DE3) / pET-21a(+)-eGFP-SUMO-FGF21 cells after enzyme digestion. The size of these bands corresponded to the expected molecular weights of eGFP-SUMO and FGF21. Meanwhile, in… Figure 4 In lane 7, two distinct bands were observed in the supernatant of the lysate after enzyme digestion of the above-mentioned bacteria, the size of which was consistent with the expected molecular weights of eGFP-SUMO and FGF21. The results indicate that the FGF21 obtained by enzyme digestion also exists mostly in a soluble form.

[0098] 1.6 Purification of FGF21

[0099] Methods: *E. coli* BL21(DE3) / pET-21a(+)-eGFP-SUMO-FGF21 fermentation cells and *E. coli* BL21(DE3) / pET-21a(+)-SUMOE fermentation cells (induction conditions as described in 1.4 above) were mixed at a wet weight ratio of 10:1 and resuspended in 20 mM PB. After high-pressure crushing and high-speed refrigerated centrifugation, the supernatant was passed through a HiTrap DEAESepharose FF (Cityva) ion exchange chromatography column to remove eGFP-SUMO protein and some other proteins. The flow-through peak was collected. The NaCl concentration of the flow-through peak solution collected from the ion exchange column was adjusted to 0.5 M, and then purified by Ni-NTA garose Resin affinity chromatography. First, the sample was washed with purification buffer D (20 mM PB, 0.5 M NaCl, 0.1 Mimidazole, pH 7.4), followed by purification buffer E (20 mM PB, 0.5 M NaCl, 0.2 Mimidazole, pH 7.4). Finally, the elution peak collected from the Ni-NTA Agarose Resin affinity chromatography column was further purified using a HiTrap Phenyl FF (High Sub) hydrophobic chromatography column (purchased from Cityva), eluted with purification buffer A (20 mM PB, pH 7.4), and the elution peak was collected for SDS-PAGE electrophoresis analysis. The results are as follows: Figure 5 As shown.

[0100] 1.7 Identification of recombinant protein FGF21 by SDS-PAGE and Western blot

[0101] Methods and Results: The purified FGF21 protein was subjected to SDS-PAGE electrophoresis, and the results are as follows: Figure 6 As shown, the protein bands are around 22 kDa, which is consistent with expectations, and the purity of FGF21 reaches 90%.

[0102] Methods and Results: Western blot analysis was performed using FGF21 monoclonal antibody (purchased from ABclonal Biotechnology). The results are as follows: Figure 7 As shown, the recombinant protein FGF21 showed a positive reaction with the FGF21 monoclonal antibody.

[0103] Example 2: Expression and purification of pET-21a(+)-eGFP-SUMO-HSA-Nb80

[0104] 2.1 Experimental Materials:

[0105] Experimental materials: pET-21a(+)-eGFP-SUMO (synthesized by a biotechnology company: the eGFP-SUMO sequence was synthesized by Nanjing Genscript Biotech Co., Ltd. and cloned into the vector pET-21a(+)), pET-21a(+)-HSA-Nb80 plasmid (synthesized by a biotechnology company: the HSA-Nb80 sequence was synthesized by Suzhou Genewise Biotech Co., Ltd. and cloned into the vector pET-21a(+)), E. coli DH5α competent cells (purchased from TransGen Biotech Co., Ltd.), E. coli BL21(DE3) competent cells (purchased from TransGen Biotech Co., Ltd.), pET-21a(+)-SUMOE plasmid (SUMO protease expression plasmid pET-21a(+)-SUMOE synthesized by Nanjing Genscript Biotech Co., Ltd.), ampicillin (Sangon Biotech Co., Ltd.), isopropyl-β-D-thiogalactoside (Guangzhou Jebes Biotechnology Co., Ltd.), HiTrap DEAE Sepharose FF (purchased from Cityva), Ni-NTA Agarose Resin (purchased from Shanghai Yisheng Biotechnology Co., Ltd.), and His-tagged monoclonal antibody (purchased from Cellsignaling).

[0106] 2.2 PCR amplification of the gene

[0107] Experimental Methods: To obtain the linearized vector of pET-21a(+)-eGFP-SUMO and the target gene fragment of HSA-Nb80 with the corresponding homologous arms, we used pET-21a(+)-eGFP-SUMO and pET-21a(+)-HSA-Nb80 plasmids as templates and performed PCR amplification of pET-21a(+)-eGFP-SUMO and HSA-Nb80 using specific primers. The amplification products were analyzed by 1.5% agarose gel electrophoresis. The primer sequences involved are shown in Table 2.

[0108] Table 2

[0109]

[0110] The eGFP-SUMO-HSA-Nb80 amino acid sequence (amino acids 1-339 are the eGFP-SUMO sequence, amino acids 340-350 (KSSHHHHHHGS) are the His tag sequence, and amino acids 351-486 are the HSA-Nb80 sequence) (SEQ ID NO.5):

[0111] VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGIEMS DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGKSSHHHHHHGSAEVQLVESGGGLVQA GGSLRLSCTASGRTFTPYTMGWFRQAPGKEREFVASILWSGNNRDYADSVKGRFAISRDNAKNTAYLQMTSLKPEDTAVYYCAAGDGLGFYRSVNQYDYWGQGTQVTVSEPKTPKGGCGGG;

[0112] eGFP-SUMO-HSA-Nb80 nucleotide sequence (the first 1017 bp of the sequence is the eGFP-SUMO gene sequence, and the last 1053-1458 bp of the sequence is the eGFP-SUMO gene sequence) (SEQ ID NO. 6):

[0113]

[0114] Result: As Figure 8 The results showed that lane 1 contained the pET-21a(+)-eGFP-SUMO linear vector, and lane 2 contained HSA-Nb80 containing the homologous arm of the pET-21a(+)-eGFP-SUMO linear vector, located at 6383 bp and 438 bp, respectively, which is consistent with the expected fragment size.

[0115] 2.3 Validation of positive clones:

[0116] Methods: Homologous recombination technology was used to ligate the amplified pET-21a(+)-eGFP-SUMO vector fragment with the HSA-Nb80 target gene fragment (containing the homologous arm of the pET-21a(+)-eGFP-SUMO linear vector) to obtain the pET-21a(+)-eGFP-SUMO-HSA-Nb80 recombinant plasmid (plasmid map shown in Figure 1). Figure 9 (as shown in (A)), and then transformed into E. coli DH5α competent cells. Single colonies were then picked from solid LB plates containing ampicillin (100 μg / mL) resistance and identified by colony PCR using specific primers for amplifying HSA-Nb80 (Table 2).

[0117] Results: The PCR products were analyzed by 1.5% agarose gel electrophoresis, and the results are as follows. Figure 9 As shown in (B), all selected monoclonal antibodies contained positive recombinants, and the amplified bands were consistent with the expected size.

[0118] 2.4 Expression analysis of the fusion protein HSA-Nb80-FGF21

[0119] Methods: *E. coli* BL21(DE3) containing the correctly sequenced pET-21a(+)-eGFP-SUMO-HSA-Nb80 recombinant plasmid were inoculated into LB broth (containing 100 μg / mL ampicillin) and cultured overnight at 37 ℃ and 220 rpm. The next day, the overnight culture was transferred 1:100 (volume / volume) to fresh LB broth (containing 100 μg / mL ampicillin). When the culture reached the logarithmic growth phase, a small amount of the culture was collected as a pre-induction sample. IPTG was added to the remaining culture to induce expression. After induction at 37 ℃ for 4 h, the culture was collected by centrifugation as a post-induction sample. The pre-induction sample was used as a control, and SDS-PAGE electrophoresis was performed.

[0120] Results: 12% SDS-PAGE electrophoresis showed that the recombinant protein eGFP-SUMO-HSA-Nb80 had a distinct band around 54 kDa in lanes 2-5. Figure 10 And all of them are consistent with the expected relative molecular weight.

[0121] 2.5 Solubility and Enzyme Digestion Analysis of Recombinant Protein eGFP-SUMO-HSA-Nb80

[0122] Methods: Induced E. coli BL21(DE3) cells containing the recombinant plasmid pET-21a(+)-eGFP-SUMO-HSA-Nb80 (named E. coli BL21(DE3) / pET-21a(+)-eGFP-SUMO-HSA-Nb80) were resuspended, homogenized using a high-pressure homogenizer, and centrifuged at 12000 rpm at 4 ℃. SDS-PAGE electrophoresis was performed on pre-induction, post-induction, post-induction, and post-induction lysate samples, as well as the supernatant and precipitate after centrifugation of the post-induction lysate, to determine the major expression form of the target protein before enzyme digestion. Meanwhile, another portion of induced E. coli BL21(DE3) / pET-21a(+)-eGFP-SUMO-HSA-Nb80 cells and induced E. coli BL21(DE3) / pET-21a(+)-SUMOE cells (the pET-21a(+)-SUMOE plasmid was transformed into E. coli BL21(DE3), and the induction conditions were the same as described in 2.4 above) were mixed at a ratio of 10:1 by wet weight of bacterial sludge, resuspended, and then homogenized using a high-pressure homogenizer. After high-speed centrifugation at 4 ℃ and 12000 rpm, the enzyme digestion lysate, the supernatant of the lysate after centrifugation, and the precipitate were taken for SDS-PAGE electrophoresis analysis to determine the enzyme digestion effect and the main expression form of the target protein after enzyme digestion.

[0123] Results: SDS-PAGE electrophoresis results showed that... Figure 11 In lanes 2 and 3, a distinct protein expression band was observed around 54 kDa in both the induced E. coli BL21(DE3) / pET-21a(+)-eGFP-SUMO-HSA-Nb80 bacterial cell and cell lysate samples, consistent with the expected molecular weight of the fusion protein eGFP-SUMO-HSA-Nb80. Meanwhile, in... Figure 11 In lane 4, a clear protein expression bar was observed in the supernatant sample of the bacterial lysate around 54 kDa. Figure 11In lane 5, the lysate precipitate of the above-mentioned bacterial cells showed almost no obvious protein expression bands around 54 kDa. The results indicate that the recombinant protein eGFP-SUMO-HSA-Nb80, before enzyme digestion, was mostly present in a soluble form.

[0124] The results after enzyme digestion showed that, Figure 11 In lane 6 of the swim grout, two distinct bands were observed in the lysate sample of E. coli BL21(DE3) / pET-21a(+)-eGFP-SUMO-HSA-Nb80 cells after enzyme digestion. The size of these bands was consistent with the expected molecular weights of eGFP-SUMO and HSA-Nb80. Meanwhile, in… Figure 11 In lane 7, two distinct bands were observed in the supernatant of the lysate after enzyme digestion of the above-mentioned bacteria, the size of which was consistent with the expected molecular weights of eGFP-SUMO and HSA-Nb80. The results indicate that the HSA-Nb80 obtained by enzyme digestion also exists mostly in a soluble form.

[0125] 2.6 Purification of HSA-Nb80

[0126] Methods: E. coli BL21(DE3) / pET-21a(+)-SUMOE fermentation cells were mixed at a wet weight ratio of 10:1 (induction conditions as described in 1.4 above) and resuspended in 20 mM PB. After high-pressure crushing and high-speed refrigerated centrifugation, the supernatant was passed through a HiTrap DEAE Sepharose FF ion exchange chromatography column to remove eGFP-SUMO protein and some other proteins. The flow-through peak was collected. The NaCl concentration of the flow-through peak solution collected from the ion exchange chromatography column was adjusted to 0.5 M, and the flow-through peak was purified by Ni-NTA Agarose Resin affinity chromatography. First, the sample was washed with purification buffer D (20 mM PB, 0.5 M NaCl, 0.1 M imidazole, pH 7.4), then eluted with purification buffer E (20 mM PB, 0.5 M NaCl, 0.2 M imidazole, pH 7.4). The elution peaks were collected and analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 12 As shown.

[0127] 2.7 Identification of recombinant proteins by SDS-PAGE and Western blot

[0128] Experimental methods and results: The purified HSA-Nb80 protein was subjected to SDS-PAGE electrophoresis, and the results are as follows: Figure 13As shown, the protein band is around 15 kDa, consistent with expectations. Furthermore, the purity of Nb80 reaches 96%.

[0129] Experimental methods and results: Western blot analysis was performed using His-tagged monoclonal antibodies, and the results are as follows: Figure 14 As shown, the recombinant protein HSA-Nb80 showed a positive reaction with the His-tagged monoclonal antibody.

[0130] Example 3: Expression of pET-21a(+)-eGFP-SUMO-SAF2

[0131] 3.1 Construction of pET-21a(+)-eGFP-SUMO-SAF2 plasmid and its prokaryotic expression

[0132] 3.1.1 Multi-segment homologous recombination

[0133] (1) Extraction of pET-21a(+)-eGFP-SUMO plasmid

[0134] Extract the pET-21a(+)-eGFP-SUMO plasmid (same as in Example 1) and pET-21a(+)-SAF2 plasmid (GenScript Biotechnology Co., Ltd.) according to the Plasmid Mini Kit I (Omega) instructions.

[0135] (2) Amplification of recombinant fragments

[0136] The multi-fragment homologous recombination method was used. Amplification primers were designed according to the instructions of ClonExpress Ultra One Step Cloning KitV2 (Vazyme) (Table 3). The target gene SAF2 was amplified using pET-21a(+)-SAF2 as a template, and linearized vector fragments 1 and 2 were amplified using pET-21a(+)-eGFP-SUMO as a template.

[0137] Table 3

[0138]

[0139] The amplification reaction mixture was prepared according to the PrimeSTAR Max DNA Polymerase (TaKaRa) instructions, as shown in Table 4.

[0140] Table 4

[0141]

[0142] The amplification procedure is set as shown in Table 5:

[0143] Table 5

[0144]

[0145] (3) Identification and purification of recombinant fragments.

[0146] The amplification products were subjected to agarose gel electrophoresis, and the gel was recovered according to the instructions of the Gel Extraction Kit (Omega). The agarose gel electrophoresis procedure is as follows:

[0147] a. Gel preparation: Weigh agarose, add 1×TAE buffer, and microwave until completely melted. After slightly cooling, add Goldview nucleic acid dye (1 / 10000) and mix well.

[0148] b. Glue pouring: Pour the glue into the glue-making tank with the comb inserted, and let it stand at room temperature until it is completely solidified.

[0149] c. Sample loading: Remove the comb, place the gel into the electrophoresis tank, and pour in buffer to cover the gel surface. Mix the DNA sample with 6×DNA loading buffer, then add the mixture sequentially to the wells and spot the DNA marker.

[0150] d. Electrophoresis: Cover the tank, turn on the power, and set the voltage to 80 V. Stop electrophoresis when the indicator has migrated to about 2 / 3 of the gel.

[0151] e. Imaging: Remove the gel, place it under a UV gel imaging system to observe the bands, and take photos for later use.

[0152] (4) Recombining fragment links

[0153] The purified target gene SAF2 and linearized vector fragments 1 and 2 obtained from amplifying pET-21a(+)-eGFP-SUMO were ligated to construct the recombinant plasmid pET-21a(+)-eGFP-SUMO-SAF2; the program was: 50℃, 15 min; then cooled to 4℃ or immediately placed on ice.

[0154] The amino acid sequence of eGFP-SUMO-SAF2 is as follows (the first amino acid (M) is the promoter amino acid sequence, amino acids 2-340 are the eGFP-SUMO sequence, and amino acids 341-576 are the SAF2 sequence; the SUMO protease cleavage site is located at the junction of eGFP-SUMO (positions 1-340) and SAF2 (positions 341-576), i.e., between amino acids 340 and 341, achieving seamless cleavage) (SEQ ID NO.7):

[0155] MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGIEMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGMPLPPHPGHPGYINFSYEVLTPLKWYQSIRPPGGGGSGGGRGSMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKSGRGGGSGGGGSPLPPMLPDLTLEAWPSTDKTKREEVD;

[0156] The nucleotide sequence of eGFP-SUMO-SAF2 is as follows (the 1st to 3rd bp (ATG) in the sequence is the promoter sequence, the 4th to 1020th bp is the eGFP-SUMO gene sequence, and the 1021st to 1728th bp is the SAF2 sequence) (SEQ ID NO.8):

[0157]

[0158] 3.1.2 Transformation of recombinant plasmid into DH5α

[0159] The constructed pET-21a(+)-eGFP-SUMO-SAF2 plasmid was transformed into DH 5α competent cells of the cloned strain, as detailed below:

[0160] ① Thawing on ice: Remove competent cells from -80℃ and plasmids (or ligation products) from -20℃ and thaw them on ice;

[0161] ② Add plasmid: Add plasmid (volume ≤ 1 / 10 of competent state volume) to competent cells, gently stir with pipette tip, and then incubate on ice for 30 min;

[0162] ③ Heat shock treatment: Heat shock in a 42℃ water bath for 45 seconds, then immediately ice bath for 2 minutes, and let stand;

[0163] ④ Resuscitation culture: Add LB medium and gently mix by pipetting. Incubate at 37℃ with shaking at 220 rpm for 1 h;

[0164] ⑤ Spread culture: Centrifuge at 5000 rpm for 5 min, discard part of the supernatant and resuspend the bacterial cells, then spread an appropriate amount onto LB plates.

[0165] 3.1.3 Colony PCR, Sequencing, and Preservation

[0166] Single colonies were picked from the transformation plate and amplified using 2× Rapid Taq Master Mix (Vazyme). The amplification products were verified by agarose gel electrophoresis. Single colonies with correct electrophoresis results were sent for sequencing. Single colonies with correct sequencing results were amplified and cultured, and then mixed with 60% sterile glycerol and LB bacterial solution at a 1:1 (v / v) ratio and stored at -80℃.

[0167] 3.1.4 Strain activation and pET-21a(+)-eGFP-SUMO-SAF2 plasmid extraction

[0168] The plasmid pET-21a(+)-eGFP-SUMO-SAF2 was extracted according to the Plasmid Mini Kit I (Omega) instructions.

[0169] 3.1.5 Transformation of BL 21 (DE 3) with recombinant plasmid

[0170] The extracted pET-21a(+)-eGFP-SUMO-SAF2 plasmid was transformed into competent cells BL 21(DE 3).

[0171] 3.1.6 Induced expression of eGFP-SUMO-SAF2 and SUMOE

[0172] In a sterile laminar flow hood, single colonies of successfully transformed BL 21(DE 3) were picked from LB agar plates and inoculated into 5 mL of Amp-resistant (1:1000) LB medium in test tubes. The colonies were incubated overnight (12-14 h) at 37°C and 220 rpm with shaking. 1% (v / v) of the overnight colonies were inoculated into 250 mL of Amp-resistant LB medium in shake flasks and amplified at 37°C and 220 rpm for 2 h (OD value reached 0.6-0.8). One mL of the amplified bacterial culture before induction was stored at -20°C. Then, 0.1 mmol / L, 0.5 mmol / L, and 1 mmol / L IPTG inducer were added to separate shake flasks, and induction was performed at 150 rpm for 4 h. Simultaneously, BL 21(DE 3) strains transformed with plasmid pET-21a(+)-SUMOE were activated and induced to express under the same conditions.

[0173] 3.1.7 Homogenizer Sterilization

[0174] The bacteria were lysed using a microfluidic homogenizer. The BL21(DE 3) bacterial suspension expressing eGFP-SUMO-SAF2 and SUMOOE were lysed together at a mass ratio of 1:10 to cleave eGFP-SUMO-SAF2 and obtain the target protein SAF2.

[0175] 3.2 Results

[0176] 3.2.1 Amplification of homologous recombination fragments

[0177] pET-21a(+)-eGFP-SUMO-SAF2 was constructed using a multi-fragment homologous recombination method, as follows: Figure 15 As shown, the target gene SAF2 (lane 1) was amplified using pET-21a(+)-SAF2 as a template, with a size of 737 bp. Using pET-21a(+)-eGFP-SUMO as a template, linearized vector fragments 1 and 2 (lanes 2 and 3) were amplified, with sizes of 2608 bp and 3783 bp, respectively. The sizes of the electrophoretic bands were consistent with expectations.

[0178] 3.2.2 Colony PCR Identification

[0179] Five positive monoclonal strains were selected from the ligation transformation plate. Using the colonies of the pET-21a(+)-eGFP-SUMO-SAF2 recombinant plasmid as templates, the amplified fragment was approximately 729 bp in size. Figure 16 As shown, the band size is consistent with expectations. The designed forward and reverse primers are located on the target gene and the linearized vector, respectively, indicating that the target gene and the linearized vector gene sequences have been successfully ligated.

[0180] 3.2.3 Expression Analysis

[0181] The eGFP-SUMO-SAF2 fusion protein is 63.8 kDa in size. SAF2 is fused with the lysing tag SUMO, and eGFP-SUMO can be removed by SUMOE cleavage. Figure 17 As shown, different concentrations of IPTG did not significantly affect the expression of eGFP-SUMO-SAF2. The pET-21a(+)-eGFP-SUMO-SAF2 plasmid expressed SAF2 in mostly soluble form, but at very low levels.

[0182] Example 4: Expression of the pET-21a(+)-eGFP-SUMO-SAF2 mutant

[0183] 4.1 Experimental Materials:

[0184] ① pET-21a(+)-eGFP-SUMO plasmid and pET-21a(+)-SUMOE plasmid, same as in Example 1;

[0185] ② pET-21a(+)-SAF2 mutant plasmid: First, replace the two cysteine ​​(C) residues at positions 121 and 139 in SAF2 with serine (S) to obtain the SAF2 mutant. Then, construct the pET-21a(+)-SAF2 mutant plasmid using the same method as in Example 3.

[0186] ③ Reagent preparation:

[0187] Equilibration buffer: 2 mmol / L EDTA (ethylenediaminetetraacetic acid), 5 mmol / L DTT (dithiothreitol), basal buffer 50 mmol / L PB, pH: 7.5;

[0188] Elution buffer: 2 mmol / L EDTA, 5 mmol / L DTT, 2 mol / L NaCl, basal buffer 50 mmol / L PB, pH: 7.5.

[0189] 4.2 Plasmid construction and prokaryotic expression of the pET-21a(+)-eGFP-SUMO-SAF2 mutant

[0190] 4.2.1 Multi-segment homologous recombination

[0191] (1) Extraction of pET-21a(+)-eGFP-SUMO plasmid

[0192] Extract the pET-21a(+)-eGFP-SUMO plasmid and the pET-21a(+)-SAF2 mutant plasmid according to the Plasmid Mini Kit I (Omega) instructions.

[0193] (2) Amplification of recombinant fragments

[0194] The multi-fragment homologous recombination method was used. Amplification primers were designed according to the instructions of ClonExpress Ultra One Step Cloning KitV2 (Vazyme) (Table 6). The SAF2 mutant was amplified using pET-21a(+)-SAF2 as a template, and linearized vector fragments 1 and 2 were amplified using pET-21a(+)-eGFP-SUMO as a template.

[0195] Table 6

[0196]

[0197] The amplification reaction mixture was prepared according to the PrimeSTAR Max DNA Polymerase (TaKaRa) instructions as shown in Table 7:

[0198] Table 7

[0199]

[0200] The amplification procedure is set as shown in Table 8:

[0201] Table 8

[0202]

[0203] (3) Identification and purification of recombinant fragments.

[0204] The method is the same as 3.1.1 in Example 3 above.

[0205] (4) Recombining fragment links

[0206] The purified target gene SAF2 mutant and linearized vector fragments 1 and 2 obtained by amplifying pET-21a(+)-eGFP-SUMO were ligated, and the reaction system is shown in Table 9.

[0207] Table 9

[0208]

[0209] Note: X and Y are determined by the molar ratio of vector to insert fragment. For single-fragment homologous recombination, the ratio is 1:2, and for multi-fragment homologous recombination, the ratio is 1:1.

[0210] Program: 50°C, 15 min; cool to 4°C or immediately on ice.

[0211] The amino acid sequence of the eGFP-SUMO-SAF2 mutant is as follows (the first amino acid (M) is the promoter amino acid sequence, amino acids 2-340 are the eGFP-SUMO sequence, and amino acids 341-576 are the mutant SAF2 sequence); amino acids 461 and 479 are mutation sites: the two cysteine ​​residues (C) are replaced with serine residues (S) respectively (SEQ ID NO.9):

[0212] ;

[0213] The nucleotide sequence of the eGFP-SUMO-SAF2 mutant is as follows (the first 3 bp (ATG) is the promoter sequence, the 4th-1020th bp is the eGFP-SUMO gene sequence, and the 1021st-1728th bp is the mutant SAF2 sequence; the 1382nd base is mutated to "C", the 1436th base is mutated to "C", and the 1437th base is mutated to "T") (SEQ ID NO.10):

[0214]

[0215] 4.2.2 Transformation of recombinant plasmid into DH5α

[0216] The constructed pET-21a(+)-eGFP-SUMO-SAF2 mutant plasmid was transformed into DH 5α competent cells of the cloned strain, using the same method as in 3.1.2 of Example 3 above.

[0217] 4.2.3 Colony PCR, Sequencing, and Preservation

[0218] Single colonies were picked from the transformation plate and amplified using 2 × Rapid Taq Master Mix (Vazyme). The amplification products were verified by agarose gel electrophoresis. Single colonies with correct electrophoresis results were sent for sequencing. Single colonies with correct sequencing results were amplified and cultured, and then mixed with 60% sterile glycerol and LB bacterial solution at a 1:1 ratio and stored at -80℃.

[0219] 4.2.4 Strain activation and extraction of eGFP-SUMO-SAF2 mutant plasmid

[0220] Extract the pET-21a(+)-eGFP-SUMO-SAF2 mutant plasmid according to the Plasmid Mini Kit I instructions.

[0221] 4.2.5 Transformation of recombinant plasmid into BL 21 (DE 3)

[0222] The extracted pET-21a(+)-eGFP-SUMO-SAF2 mutant plasmid was transformed into competent cells BL 21(DE3).

[0223] 4.2.6 Induced expression of eGFP-SUMO-SAF2 mutant and SUMOE

[0224] In a sterile laminar flow hood, successfully transformed BL 21(DE 3) single colonies were picked from LB agar plates and inoculated into 5 mL of Amp-resistant (1:1000) LB medium in test tubes. The colonies were incubated overnight (12-14 h) at 37°C and 220 rpm with shaking. 1% of the overnight colonies were then inoculated into 250 mL of Amp-resistant LB medium in shake flasks and amplified at 37°C and 220 rpm for 2 h (OD value reached 0.6-0.8). One mL of the amplified bacterial culture before induction was stored at -20°C. Then, 0.1 mmol / L, 0.5 mmol / L, and 1 mmol / L IPTG inducer were added to separate shake flasks, and induction was performed at 150 rpm for 4 h. Simultaneously, BL 21(DE 3) strains transformed with plasmid pET-21a(+)-SUMOE were activated and induced to express under the same conditions.

[0225] 4.2.7 Homogenizer Sterilization

[0226] The bacteria were lysed using a microfluidic homogenizer. The BL 21(DE 3) bacterial suspension expressing eGFP-SUMO-SAF2 mutant and SUMOE were lysed together at a mass ratio of 1:10 to cleave the eGFP-SUMO-SAF2 mutant and obtain the target protein SAF2 mutant.

[0227] 4.3 Results

[0228] 4.3.1 Amplification of homologous recombination fragments

[0229] The pET-21a(+)-eGFP-SUMO-SAF2 mutant was constructed using a multi-fragment homologous recombination method, such as... Figure 18 As shown, the target gene SAF2 mutant (lane 1) was amplified using pET-21a(+)-SAF2 mutant as a template, with a size of 737 bp. Using pET-21a(+)-eGFP-SUMO as a template, linearized vector fragments 1 and 2 (lanes 2 and 3) were amplified, with sizes of 2608 bp and 3783 bp, respectively. The sizes of the electrophoretic bands were consistent with expectations.

[0230] 4.3.2 Colony PCR Identification

[0231] Five positive monoclonal strains were selected from the ligation transformation plate. Using the colonies of the pET-21a(+)-eGFP-SUMO-SAF2 mutant recombinant plasmid as templates, the amplified fragment was approximately 729 bp in size. Figure 19 As shown, the band size is consistent with expectations. The designed forward and reverse primers are located on the target gene and the linearized vector, respectively, indicating that the target gene and the linearized vector gene sequences have been successfully ligated.

[0232] 4.3.3 Expression Analysis

[0233] like Figure 20 As shown in Figure A, the green fluorescence of the bacterial culture indicates successful expression of the eGFP-SUMO-SAF2 mutant fusion protein. Furthermore, among the four centrifuge tubes, tube 3 exhibits stronger fluorescence than tube 4, suggesting that the fusion protein is primarily expressed in soluble form. Figure 20 As shown in B, fusing the SAF2 mutant with eGFP-SUMO yields a highly expressed and mostly soluble eGFP-SUMO-SAF2 mutant.

[0234] 4.3.4 Separation and Purification

[0235] The eGFP-SUMO-SAF2 mutant was isolated and purified after SUMOE digestion. The molecular weight and isoelectric point of the SAF2 mutant were predicted to be 25.5 kDa and 9.57, respectively, using the ProtParam online tool. The equilibration buffer and elution buffer used for isolation and purification were both pH 7.5. pH 7.5 is close to the optimal pH for SUMOase activity, which is beneficial for the cleavage of the eGFP-SUMO-SAF2 mutant.

[0236] CM ion exchange chromatography and HP affinity chromatography:

[0237] (1) Sample and buffer preparation: The sample buffer needs to be filtered through a 0.45 μm filter membrane. Other solutions that need to flow through the column, including deionized water, equilibration buffer, elution buffer, alcohol, etc., also need to be filtered to prevent impurities from clogging the column.

[0238] (2) Install the chromatography column: Connect the appropriate chromatography column HiTrap CM FF 5 mL (Cytiva) or HiTrap HeparinHP 5 mL (Cytiva) to the AKTA system, ensuring the correct flow direction and avoiding air bubbles from entering.

[0239] (2) System equilibration (cleaning and equilibration): First, rinse the alcohol stored in the tubing with water until the monitored conductivity and UV baseline levels are reached, then rinse with equilibration buffer for 5 column volumes.

[0240] (3) Loading: After the baseline stabilizes, reduce the flow rate to 1-2 mL / min for loading. At this time, the target protein will bind to the ion exchange groups on the medium, while the flow-through peak records the substances that failed to bind.

[0241] (4) Washing or rebalancing: Wash away non-specific or weakly binding proteins with equilibration buffer until the UV detector signal returns to near baseline.

[0242] (5) Elution: A salt concentration (NaCl) gradient from 0% to 100% is used. As the proportion of high-salt buffer increases, competitive ions replace the target protein one by one.

[0243] (6) Collection: The separation effect can be judged based on the fluctuation of the ultraviolet absorption peak, and the components can be collected according to the changes of the ultraviolet peak.

[0244] (7) Column regeneration and preservation: After elution, thoroughly wash the column with high-salt buffer to remove all tightly bound residues. Finally, replace the salt with pure water, seal the column with 20% (V / V) ethanol to prevent microbial growth, and seal both ends of the column before removing it from the system.

[0245] (8) Detection and analysis: After SDS-PAGE electrophoresis, the collected components were detected by Coomassie brilliant blue staining.

[0246] The results are as follows Figure 21 and Figure 22 As shown: the target protein flows through the column without sticking. The SAF2 mutant flows through the column and precipitates in the heparin chromatography column.

[0247] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A visual expression system for promoting soluble expression of recombinant proteins in *E. coli*, characterized in that, It can be constructed using any of the following methods: Method 1: (1) Linearize the recombinant expression vector containing green fluorescent protein eGFP and soluble tag SUMO to obtain a linearized recombinant expression vector; (2) The target gene fragment is ligated into a linearized recombinant expression vector to obtain the Escherichia coli visual expression system that promotes the soluble expression of recombinant proteins; Method 2: (3) ① Connect the gene sequence of green fluorescent protein eGFP, the gene sequence of the dissolution tag SUMO and the target gene sequence in sequence to obtain target fragment 1; Alternatively: ② Connect the gene sequence of green fluorescent protein eGFP, the gene sequence of the dissolution tag SUMO, the His tag sequence and the target gene sequence in sequence to obtain target fragment 2; (4) Connect the target fragment 1 or the target fragment 2 to a linearized prokaryotic expression vector to obtain the Escherichia coli visualization expression system that promotes the soluble expression of recombinant proteins.

2. The Escherichia coli visualization expression system according to claim 1, characterized in that: The protein encoded by the target gene in steps (2) and (3) is at least one of FGF21, HSA-Nb80, SAF2 and SAF2 mutant; The amino acid sequence of FGF21 is shown in positions 351-545 of SEQ ID NO.3, and the nucleotide sequence is shown in positions 1053-1635 of SEQ ID NO.4; The amino acid sequence of HSA-Nb80 is shown in positions 351-486 of SEQ ID NO.5, and the nucleotide sequence is shown in positions 1053-1458 of SEQ ID NO.6; The amino acid sequence of the SAF2 is shown in positions 341-576 of SEQ ID NO.7, and the nucleotide sequence is shown in positions 1021-1728 bp of SEQ ID NO.8; The SAF2 mutant is characterized by the mutation of two cysteine ​​residues at positions 121 and 139 in SAF2 to serine residues. Its amino acid sequence is shown as positions 341-576 in SEQ ID NO.9, and its nucleotide sequence is shown as positions 1021-1728 bp in SEQ ID NO.

10.

3. The Escherichia coli visualization expression system according to claim 1, characterized in that: The amino acid sequences of the green fluorescent protein eGFP and the dissolution tag SUMO mentioned in step (1) are shown in positions 1-339 of SEQ ID NO.3, and their nucleotide sequences are shown in positions 1-1017bp of SEQ ID NO.4; The target fragment 1 mentioned in step (3) is eGFP-SUMO-SAF2 or an eGFP-SUMO-SAF2 mutant; wherein, the protein sequence encoded by the eGFP-SUMO-SAF2 is shown in SEQ ID NO.7 and the nucleotide sequence is shown in SEQ ID NO.8; the protein sequence encoded by the eGFP-SUMO-SAF2 mutant is shown in SEQ ID NO.9 and the nucleotide sequence is shown in SEQ ID NO.10; The target fragment 2 mentioned in step (3) is eGFP-SUMO-FGF21 or eGFP-SUMO-HSA-Nb80; wherein, the protein sequence encoded by eGFP-SUMO-FGF21 is shown in SEQ ID NO.3 and the nucleotide sequence is shown in SEQ ID NO.4; the protein sequence encoded by eGFP-SUMO-HSA-Nb80 is shown in SEQ ID NO.5 and the nucleotide sequence is shown in SEQ ID NO.

6.

4. The Escherichia coli visualization expression system according to claim 1, characterized in that: The amino acid sequence of the His tag mentioned in step (3) is as follows: KSSHHHHHHGS.

5. The Escherichia coli visualization expression system according to claim 1, characterized in that: The starting vector for the recombinant expression vector mentioned in step (1) is the pET-21a series vector; The prokaryotic expression vector mentioned in step (4) is the pET-21a series vector.

6. The Escherichia coli visualization expression system according to claim 5, characterized in that: The starting vector for the recombinant expression vector mentioned in step (1) is the pET-21a(+) vector; The prokaryotic expression vector mentioned in step (4) is the pET-21a(+) vector.

7. The Escherichia coli visualization expression system according to claim 1, characterized in that: The target gene fragment mentioned in step (2) is a target gene fragment with a homologous arm of a prokaryotic expression vector.

8. The Escherichia coli visualization expression system according to claim 1, characterized in that: The target gene fragment mentioned in step (2) is at least one of the following: FGF21 fragment, HSA-Nb80 fragment, SAF2 fragment, and SAF2 mutant fragment; wherein, The FGF21 fragment was amplified using pUC-GW-Kan-FGF21 plasmid as a template and primers SEQ ID NO.13 and SEQ ID NO.

14. The HSA-Nb80 fragment was amplified using pET-21a(+)-HSA-Nb80 plasmid as a template and primers SEQ ID NO.15 and SEQ ID NO.

16. The SAF2 fragment was amplified using pET-21a(+)-SAF2 plasmid as a template and primers SEQ ID NO.17 and SEQ ID NO.

18. The SAF2 mutant fragment was amplified using the pET-21a(+)-SAF2 mutant plasmid as a template and primers SEQ ID NO.23 and SEQ ID NO.

24.

9. The application of the Escherichia coli visualization expression system for promoting soluble expression of recombinant proteins as described in any one of claims 1 to 8 in the soluble expression of recombinant proteins.

10. The application according to claim 9, characterized in that: The recombinant protein includes at least one of FGF21, HSA-Nb80, SAF2, or a SAF2 mutant; The amino acid sequence of FGF21 is shown in positions 351-545 of SEQ ID NO.3, and the nucleotide sequence is shown in positions 1053-1635 of SEQ ID NO.4; The amino acid sequence of HSA-Nb80 is shown in positions 351-486 of SEQ ID NO.5, and the nucleotide sequence is shown in positions 1053-1458 of SEQ ID NO.6; The amino acid sequence of the SAF2 is shown in positions 341-576 of SEQ ID NO.7, and the nucleotide sequence is shown in positions 1021-1728 bp of SEQ ID NO.8; The SAF2 mutant is characterized by the mutation of two cysteine ​​residues at positions 121 and 139 in SAF2 to serine residues. Its amino acid sequence is shown as positions 341-576 in SEQ ID NO.9, and its nucleotide sequence is shown as positions 1021-1728 bp in SEQ ID NO.10.

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