Composite protein, recombinant plasmid, recombinant escherichia coli and application

Abstract

The application provides a kind of composite protein, recombinant plasmid, recombinant escherichia coli and application.The composite protein includes nanometer compartment protein and the cargo protein loaded in nanometer compartment protein, nanometer compartment protein loads cargo protein through targeting peptide, the amino acid sequence of targeting peptide is as shown in Seq.ID No.1.The application first clearly defines DyP-linker as the key signal sequence of CFP-29 encapsulating cargo protein, by accurately retaining the core sequence, avoid the problem of blind modification caused by unclear signal sequence in the prior art.Target protein realizes targeted encapsulation through the specific interaction of DyP-linker and CFP-29 spherical shell protein, effectively solves the defects of low encapsulation efficiency and non-specific binding in traditional modification, ensures that the target protein can efficiently and stably enter the nanosphere shell.

Description

Technical Field

[0001] This invention relates to the field of protein modification technology, and in particular to a complex protein, a recombinant plasmid, a recombinant Escherichia coli, and their applications. Background Technology

[0002] Nanocompartment proteins are a class of functional proteins with self-assembly properties. They can spontaneously form a structurally stable icosahedral shell structure, creating a closed nanoscale cavity inside. This unique "shell-cavity" structure endows them with natural biomolecular encapsulation and protection capabilities. It not only prevents the encapsulated cargo protein from being degraded and interfered with by external factors such as proteases and reactive oxygen species, but also shows great application potential in fields such as biocatalysis, drug delivery, and biosensing.

[0003] Mycobacterium tuberculosis, as a pathogenic microorganism with a special survival mechanism, has a nanocompartment protein CFP-29 that is formed by the self-assembly of 29kDa subunits into an icosahedral structure. In its natural state, it can specifically encapsulate DyP-type peroxidase, and this characteristic provides a natural template for its development as a bio-nanocarrier.

[0004] However, the applications of naturally occurring nanocompartment proteins are limited, necessitating artificial modification that relies on the stability of the outer shell protein to encapsulate the desired functional protein. In existing CFP-29 nanocompartment protein modification technologies, the targeted binding mechanism between the cargo protein and the shell protein remains unclear. Most modification schemes rely on random screening or non-specific fusion, resulting in low encapsulation efficiency and poor encapsulation specificity of the cargo protein, and are prone to misfolding of the target protein and inability to be purified in tandem with the shell. Furthermore, some modification schemes retain the complete natural cargo protein sequence to preserve encapsulation function, which not only occupies the limited space inside the shell but may also introduce unnecessary enzyme activity interference, further limiting its adaptability to various applications. Summary of the Invention

[0005] The purpose of this invention is to address the problems of low encapsulation efficiency and low adaptability of CFP-29 nanocompartment protein in the prior art, and to provide a composite protein, recombinant plasmid, recombinant Escherichia coli and its applications.

[0006] The technical solution adopted to achieve the purpose of this invention is: In a first aspect, the present invention provides a composite protein comprising a nanocompartment protein and a cargo protein loaded within the nanocompartment protein. The cargo protein is loaded by a targeting peptide, the amino acid sequence of which is shown in Seq. ID No. 1. Specifically, the cargo protein can be selectively targeted by a C-terminal sequence, specifically targeted and encapsulated within the nanocompartment protein. This selective C-terminal sequence is the targeting peptide DyP-linker of the present invention.

[0007] In some alternative embodiments, the cargo protein includes one or more of the following: tyrosine recombinase Flp, peroxidase Dyp, superoxide dismutase SOD, green fluorescent protein GFP, folic acid biosynthesis protein FolB, bacterial ferritin Bfr, ​​ferricredoxin Fd, iron mineralization active encapsulation protein associated Firmicutes protein IMEF, nitrite reductase domain and hydroxylamine oxidoreductase domain fusion protein NIR-HAO, earthworm-like hemoglobin, erythroglobin, and biphenyl dehydrogenase protein.

[0008] In some alternative embodiments, the cargo protein includes superoxide dismutase (SOD) and / or green fluorescent protein (GFP).

[0009] In some alternative embodiments, the diameter of the nanocompartment protein is 24 nm to 42 nm.

[0010] In some alternative embodiments, the composite protein satisfies at least one of the following: (1) the composite protein has greater resistance to enzymatic cleavage than the cargo protein; (2) the composite protein has greater stability at 25°C to 65°C than the cargo protein; and (3) the composite protein has higher stability in an environment with a pH of 3 to 12 than the cargo protein.

[0011] In a second aspect, the present invention provides a recombinant plasmid for expressing the complex protein of the first aspect, the recombinant plasmid comprising the sequence shown in Seq. ID No. 3.

[0012] In some alternative implementations, the recombinant plasmid includes a sequence as shown in Seq.ID No.4.

[0013] Thirdly, the present invention provides a recombinant Escherichia coli comprising the recombinant plasmid of the second aspect and / or secreting the complex protein of the first aspect.

[0014] Fourthly, the present invention provides the application of a composite protein of the first aspect in enzyme catalysis, cell engineering, imaging detection, or fluorescent labeling.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention, for the first time, clearly identifies DyP-linker as a key signal sequence for CFP-29 encapsulating cargo proteins. By precisely preserving this core sequence, it avoids the problem of blind modification caused by unclear signal sequences in existing technologies. The target protein achieves targeted encapsulation through the specific interaction between DyP-linker and CFP-29 shell protein, effectively solving the defects of low encapsulation efficiency and excessive non-specific binding in traditional modifications, ensuring that the target protein can enter the interior of the nanospheres efficiently and stably.

[0016] 2. This invention, by knocking out the non-essential sequence of DyP-type peroxidase and retaining only the key linker sequence mediating encapsulation, provides ample space and adaptability for the insertion of target proteins. Whether it is a small functional protein, an enzyme molecule, or a diagnostic marker protein, all can achieve targeted binding to the CFP-29 shell by inserting their DNA sequence. This breaks the limitation of natural CFP-29 encapsulating only a single cargo protein, allowing the nanocompartment to flexibly load various target proteins according to different application requirements, greatly expanding its application boundaries.

[0017] 3. The CFP-29 protein itself possesses a stable spherical shell structure and excellent purification properties. In this invention, the target protein forms a stable complex with the CFP-29 spherical shell via the DyP-linker, enabling simultaneous purification using the mature CFP-29 purification system. This "shell-target protein" synergistic purification mode avoids the problems of easy degradation and denaturation of the target protein in traditional protein purification. Attached Figure Description

[0018] Figure 1 The image shows a schematic diagram of the raw material plasmid.

[0019] Figure 2 The image shows a schematic diagram of the recombinant SOD plasmid.

[0020] Figure 3 The image shows the elution curve of the SOD complex protein chromatographic column.

[0021] Figure 4 The image shown is an SDS-PAGE diagram of the SOD complex protein.

[0022] Figure 5 The image shown is a negative-stained electron microscope image of the SOD complex protein.

[0023] Figure 6 The image shows a schematic diagram of the recombinant GFP plasmid.

[0024] Figure 7 The image shows the elution curve of the GFP complex protein column.

[0025] Figure 8The image shown is an SDS-PAGE diagram of the GFP complex protein.

[0026] Figure 9 The image shown is a negative-stained electron micrograph of the GFP complex protein.

[0027] Figure 10 The image shown is a physical representation of the GFP complex protein.

[0028] Figure 11 The diagram shown is a schematic of the recombinant plasmid of Comparative Example 1.

[0029] Figure 12 The figure shows the elution curve of the composite protein column in Comparative Example 1.

[0030] Figure 13 The image shown is an SDS-PAGE diagram of the complex protein in Comparative Example 1.

[0031] Figure 14 The image shows the results of a test on the ability of SOD to reduce cellular oxidative stress.

[0032] Figure 15 The image shows the results of an Enc (SOD) test to reduce cellular oxidative stress.

[0033] Figure 16 The image shown is an SDS-PAGE image of SOD digested by trypsin.

[0034] Figure 17 The image shown is an SDS-PAGE image of Enc (SOD) after trypsin digestion.

[0035] Figure 18 The image shows SDS-PAGE images of Enc (SOD) after incubation at different temperatures.

[0036] Figure 19 The image shows SDS-PAGE images of Enc (SOD) after incubation at different pH values. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0038] The amino acid sequence of the DyP-linker in this invention is Seq.ID No.1: N-terminus-GSLSIGSLKGSPR-C-terminus.

[0039] Amino acid sequence of CFP-29 protein Seq.ID No.2: N-terminal - NNLYRDLAPVTEAAWAEIELEAARTFKRHIAGRRVVDVSDPGGPVTAAVSTGRLIDVKAPTNGVIAHLRASKPLVRLRVPFTLSRNEIDDVERGSKDSDWEPVKEAAKKLAFVEDRTIFEGYSAASIEGIRSASSNPALTLPEDPREIPDVISQALSELRLAGVDGPYSVLLSADVYTKVSETSDHGYPIREHLNRLVDGDIIWAPAIDGAFVLTTRGGDFDLQLGTDVAIGYASHDTDTVRLYLQETLTFLCYTAEASVALSH - C-terminal.

[0040] DNA sequence expressing DyP-linker Seq.ID No.3: 3’-GGGTCGCTATCGATCGGCAGCTTGAAAGGAAGCCCCCGATGA-5’.

[0041] The DNA sequence expressing CFP-29 protein Seq.ID No.4: 3’-AACAATCTCTACCGCGATTTGGCACCGGTCACCGAAGCCGCTTGGGCGGAAATCGAATTGGAGGCGGCGCGGACGTTCAAGCGACACATCGCCGGGCGCCGGGTGGTCGATGTCAGTGATCCCGGGGGGCCCGTCACCGCGGCGGTCAGCACCGGCCGGCTGATCGATGTTAAGGCACCAACCAACGGCGTGATCGCCCACCTGCGGGCCAGCAAACCCCTTGTCCGGCTACGGGTTCCGTTTACCCTGTCGCGCAACGAGATCGACGACGTGGAACGTGGCTCTAAGGACTCCGATTGGGAACCGGTAAAGGAGGCGGCCAAGAAGCTGGCCTTCGTCGAGGACCGCACAATATTCGAAGGCTACAGCGCCGCATCAATCGAAGGGATCCGCAGCGCGAGTTCGAACCCGGCGCTGACGTTGCCCGAGGATCCCCGTGAAATCCCTGATGTCATCTCCCAGGCATTGTCCGAACTGCGGTTGGCCGGTGTGGACGGACCGTATTCGGTGTTGCTCTCTGCTGACGTCTACACCAAGGTTAGCGAGACTTCCGATCACGGCTATCCCATCCGTGAGCATCTGAACCGGCTGGTGGACGGGGACATCATTTGGGCCCCGGCCATCGACGGCGCGTTCGTGCTGACCACTCGAGGCGGCGACTTCGACCTACAGCTGGGCACCGACGTTGCAATCGGGTACGCCAGCCACGACACGGACACCGTGCGCCTCTACCTGCAGGAGACGCTGACGTTCCTTTGCTACACCGCCGAGGCGTCGGTCGCGCTCAGCCAC-5’。

[0042]

[0043] SOD gene sequence Seq.ID No.6: 3'-ATGGCGCCGTCCGGGGAAGACCACGGTGGCGGGCACGGTGCCGGCGCGGCAGGCGCCGGTGAGACCCTCACCGCAGAACTCAAGACCGCCGACGGAACCTCGGTGGCAACCGCCGACTTCCAGTTCGCCGACGGCTTTGCCACGGTGACGATCGAGACCACCACCCCGGGTCGCCTCACCCCCGGCTTCCACGGTGTGCACATTCACTCGGTGGGCAAGTGCGAGGCGAACTCCGTCGCCCCGACCGGTGGCGCGCCCGGTGACTTCAACTCCGCCGGCGGCCACTTCCAGGTGTCCGGCCACAGCGGACATCCCGCCAGCGGCGACCTGAGCTCGCTGCAGGTCCGGGCCGACGGCTCGGGCAAGCTGGTGACCACCACCGATGCGTTCACGGCCGAGGATCTGCTCGACGGCGCCAAGACCGCGATCATCATCCACGAGAAGGCCGACAACTTCGCCAACATCCCGCCGGAGCGCTACCAGCAGGTCAACGGCGCACCCGGCCCGGATCAGACGACGATGGCCACCGGCGACGCCGGAAGTCGGGTGGCGTGCGGTGTCATCTCTGCCGGC-5'.

[0044] SOD amino acid sequence Seq.ID No.7: N-terminus - MAPSGEDHGGGHGAGAAGAGETLTAELKTADGTSVATADFQFADGFATVTIETTTPGRLTPGFHGVHIHSVGKCEANSVAPTGGAPGDFNSAGGHFQVSGHSGHPASGDLSSLQVRADGSGKLVTTTDAFTAEDLLDGAKTAIIIHEKADNFANIPPERYQQVNGAPGPDQTTMATGDAGSRVACGVISAG - C-terminus.

[0045] GFP gene sequence Seq.ID No.8: 3’-ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAA-5’。

[0046] GFP amino acid sequence Seq.ID No.9: N-terminal-MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGD TLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK-C terminus.

[0047] Example 1 A complex protein Enc (SOD) comprising CFP-29 protein and SOD enzyme loaded within CFP-29 protein.

[0048] This complex protein was prepared using the following method: Step 1, construct plasmid: By such Figure 1 The DyP gene sequence was knocked out in the raw material plasmid shown, while the DyP-linker gene sequence, the target peptide at the C-terminus of DyP-type peroxidase, was retained. An SOD gene sequence was inserted at the original DyP gene sequence location. This small peptide linked to the cargo protein SOD and autonomously loaded SOD into the nanocompartment protein. The plasmid was then reconstructed, as shown below. Figure 2 As shown in the figure. The DyP gene sequence is shown in Seq.ID No. 5, the SOD gene sequence is shown in Seq.ID No. 6, and the SOD amino acid sequence is shown in Seq.ID No. 7.

[0049] Step 1.1, Obtain the linear template: The PCR system (50 μL) was constructed as follows: 1 μL of raw material plasmid, 1 μL each of primers duet1-R and duet1-F, 1 μL of DNA polymerase, 1 μL of DMSO, 1 μL of dNTPs, 19 μL of double-distilled water, and 25 μL of 2x buffer. PCR was performed using a PCR instrument. Duet1-R and duet1-F are shown below.

[0050] duet1-R: 5'-gcccatggtatatctccttcttaaagttaaacaaaattat-3'.

[0051] duet1-F: 5'-GGGTCGCTATCGATCGGCAGCTTGAAAGGAAGCCCCCGAT-3'.

[0052] Step 1.2, obtain the SOD gene with homologous arms: The PCR system (50 μL) was constructed as follows: 1 μL of original plasmid, 1 μL each of primers SOD-R and SOD-F, 1 μL of DNA polymerase, 1 μL of DMSO, 1 μL of dNTPs, 19 μL of double-distilled water, and 25 μL of 2x buffer. PCR was performed using a PCR instrument. The sequences of SOD-R and SOD-F are shown below.

[0053] SOD-R: 5'-gaaggagatataccatgggcATGGCGCCGTCCGGGGAAGACCACGGTGGCGGGCACGGT-3'.

[0054] SOD-F: 5'-gaaggagatataccatgggcATGGCGCCGTCCGGGGAAGACCACGGTGGCGGGCACGGT-3'.

[0055] Step 1.3, Construction of recombinant plasmid: Constructing the recombination system (30 μL): Linear template: 0.02 × fragment length (bp) = required fragment mass (ng). Calculate the volume required based on the PCR product concentration.

[0056] SOD with homologous arms: 0.04 × fragment length (bp) = required fragment mass (ng). Calculate the volume dosage based on the PCR product concentration.

[0057] Add 2×CE recombinase and double-distilled water to a final volume of 30 μL. Incubate at 50℃ for 5 min to obtain reconstructed nanocompartment protein particles.

[0058] Step 2, Protein Expression and Purification: Step 2.1: The reconstructed nanocompartment protein particles were transformed into Escherichia coli for prokaryotic expression to obtain engineered Escherichia coli. The engineered Escherichia coli was subjected to high pressure (1000 Pa) with buffer 1 (20 mM MOPS, 100 mM NaCl, pH 7.4), and then centrifuged (18000 rpm, 40 min) to collect the supernatant.

[0059] Step 2.2: Purify the supernatant by Flag-label affinity chromatography: Load the supernatant onto a chromatographic column packed with Anti-Flag packing material, dilute the flag peptide with buffer1 to a concentration of 0.15 mg / ml, wash and elute, add the eluent to a concentration tube with a molecular weight cutoff of 100 kDa, centrifuge (3500 rpm) to less than 1 ml, and obtain the modified crude extract of E. coli nanocompartment protein encapsulated with SOD.

[0060] Step 2.3: Equilibrate the Superose 6 Increase 10 / 300 size-exclusion column with one column volume (25 mL) of buffer 1. Load the above crude extract onto the Superose 6 Increase 10 / 300 size-exclusion column and elute with 25 mL of buffer 1. The results are as follows. Figure 3 .Depend on Figure 3 It can be seen that the complex protein exhibits a characteristic peak between 10-13 ml.

[0061] Step 3, Protein Identification: Samples were collected at the characteristic peak positions to obtain a modified E. coli nanocompartment protein solution encapsulated with SOD. This solution was then analyzed by SDS-PAGE (the solution was denatured by adding 5x loading buffer and incubating in a 100°C metal bath for 1 hour). The results are as follows: Figure 4 . Figure 4 The CFP-29 site is located at approximately 29 kDa, and the SOD site is located at approximately 20 kDa. This confirms that the obtained sample is a modified E. coli nanocompartment protein encapsulated with SOD.

[0062] The modified E. coli nanocompartment protein sample encapsulating SOD was observed using a digital transmission electron microscope (120 kV). The protein concentration was 5 mg / ml, the particles were clear, and the distribution was sparse. The results are as follows. Figure 5 .

[0063] Example 2 A complex protein Enc (GFP) comprising CFP-29 protein and green fluorescent protein GFP loaded within CFP-29 protein.

[0064] This complex protein was prepared using the following method: Step 1, construct plasmid: By such Figure 1 The DyP gene sequence was knocked out in the raw material plasmid shown, while the DyP-linker gene sequence, the C-terminal targeting peptide of DyP-type peroxidase, was retained. A GFP gene sequence was inserted at the original DyP gene sequence location. This small peptide linked to GFP and autonomously loaded GFP into the nanocompartment protein. The plasmid was then reconstructed, as shown below. Figure 6 As shown in the figure. The DyP gene sequence is shown in Seq.ID No. 5, the GFP gene sequence is shown in Seq.ID No. 8, and the GFP amino acid sequence is shown in Seq.ID No. 9.

[0065] Step 1.1, Obtain the linear template: The PCR system (50 μL) was constructed as follows: 1 μL of raw material plasmid, 1 μL each of primers duet1-R and duet1-F, 1 μL of DNA polymerase, 1 μL of DMSO, 1 μL of dNTPs, 19 μL of double-distilled water, and 25 μL of 2x buffer. PCR was performed using a PCR instrument. The sequences of duet1-R and duet1-F are shown below.

[0066] duet1-R: 3'-gcccatggtatatctccttcttaaagttaaacaaaattat-5'.

[0067] duet1-F: 3'-GAGCTCAAGAAGGAGATATACCatgAACAATCTCTACCGCGATTTGGCACCGGTCACCG-5'.

[0068] Step 1.2, obtain the GFP gene with homologous arms: The PCR system (50 μL) was constructed as follows: 1 μL of raw material plasmid, 1 μL each of primers GFP-R1, GFP-R2, and GFP-F, 1 μL of DNA polymerase, 1 μL of DMSO, 1 μL of dNTPs, 18 μL of double-distilled water, and 25 μL of 2x buffer. PCR was performed using a PCR instrument. The sequences of GFP-R1, GFP-R2, and GFP-F are shown below.

[0069] GFP-R1: 3'-GGTATATCTCCTTCTTGAGCTCTCATCGGGGGCTTCCTTTCAAGCTGCCGATCGATAGC-5'.

[0070] GFP-R2: 3'-GCTTCCTTTCAAGCTGCCGATCGATAGCGACCCTTTGTAGAGCTCATCCATGCCATGT-5'.

[0071] GFP-F: 3'-agaaggagatataccatgggcATGAGCAAAGGAGAAGAACTTTTC-5'.

[0072] Step 1.3, Construction of recombinant plasmid: Constructing a recombination system (30 μL): Linear template: 0.02 × fragment length (bp) = required fragment mass (ng), calculate volume usage based on PCR product concentration. For GFP with homologous arms: 0.04 × fragment length (bp) = required fragment mass (ng), calculate the volume required based on the PCR product concentration.

[0073] Add 2×CE recombinase, bring the volume up to 30 μL with double-distilled water, and run the program at 50 °C for 5 min to obtain reconstructed nanocompartment protein particles.

[0074] Step 2, Protein Expression and Purification: Step 2.1: The reconstructed nanocompartment protein particles were transformed into Escherichia coli for prokaryotic expression to obtain engineered Escherichia coli. The engineered Escherichia coli was subjected to high pressure (1000 Pa) with buffer 1 (20 mM MOPS, 100 mM NaCl, pH 7.4), and then centrifuged (18000 rpm, 40 min) to collect the supernatant.

[0075] Step 2.2: Purify the supernatant by Flag-label affinity chromatography: Load the supernatant onto a chromatographic column packed with Anti-Flag packing material, dilute the flag peptide with buffer1 to a concentration of 0.15 mg / ml, wash and elute, add the eluent to a concentration tube with a molecular weight cutoff of 100 kDa, centrifuge (3500 rpm) to less than 1 ml, and obtain the modified crude extract of E. coli nanocompartment protein encapsulated with SOD.

[0076] Step 2.3: Equilibrate the Superose 6 Increase 10 / 300 size-exclusion column with one column volume (25 ml) of buffer 1. Load the above crude extract onto the Superose 6 Increase 10 / 300 size-exclusion column and elute with 25 ml of buffer 1. The results are as follows. Figure 7 .Depend on Figure 7 It can be seen that the complex protein Enc (GFP) has a characteristic peak between 10-14 ml.

[0077] Step 3, Protein Identification: Samples were collected at the characteristic peak positions to obtain a modified E. coli nanocompartment protein solution encapsulating GFP. This solution was then subjected to SDS-PAGE analysis (the solution was treated with 5x loading buffer and the protein was denatured in a 100°C metal bath for 1 hour). The results are as follows: Figure 8 .Depend on Figure 8 It is known that CFP-29 is located at approximately 29 kDa, and GFP is located at approximately 26 kDa. This confirms that the obtained sample is the modified E. coli nanocompartment protein Enc (GFP) encapsulated with GFP.

[0078] The modified E. coli nanocompartment protein sample encapsulating GFP was observed using a digital transmission electron microscope (120 kV). The protein concentration was 5 mg / ml, and the particles were clear and sparsely distributed. The results are as follows. Figure 9 .

[0079] The resulting high-concentration E. coli nanocompartment protein solution encapsulated with GFP obtained by concentrating the protein solution was fluorescent green, as shown in the following results. Figure 10 .

[0080] Comparative Example 1 To demonstrate the key role of the N-terminal targeting peptide DyP-linker of DyP-type peroxidase in the protein encapsulation process, an Enc(SOD) plasmid without the targeting peptide DyP-linker was constructed.

[0081] By knocking out the DyP gene sequence in the raw material plasmid, without retaining the DyP-linker gene sequence (a target peptide at the C-terminus of DyP-type peroxidase), and inserting the SOD gene sequence at the original DyP gene sequence location, the plasmid is reconstructed. Figure 11 The specific method is as follows.

[0082] Obtain the linear template: The PCR system (50 μL) was constructed as follows: 1 μL of raw material plasmid, 1 μL each of primers duet1-R and duet1-F, 1 μL of DNA polymerase, 1 μL of DMSO, 1 μL of dNTPs, 19 μL of double-distilled water, and 25 μL of 2x buffer. PCR was performed using a PCR instrument. The sequences of duet1-R and duet1-F are as follows.

[0083] duet1-R: 3'-CTTCCCCGGACGGCGCCATgcccatggtatatctccttcttaaagttaaacaaaattat-5'.

[0084] duet1-F: 3'-GAGCTCAAGAAGGAGATATACCatgAACAATCTCTACCGCGATTTGGCACCGGTCA-5'.

[0085] Obtaining the SOD gene with homologous arms: Constructing a PCR system (50 μL): 1 μL of raw material plasmid, 1 μL each of primers SOD-R and SOD-F, 1 μL of DNA polymerase, 1 μL of DMSO, 1 μL of dNTPs, 19 μL of double-distilled water, and 25 μL of 2x buffer. PCR was performed using a PCR instrument. The SOD-R and SOD-F sequences are as follows.

[0086] SOD-R: 3'-GAGATTGTTcatGGTATATCTCCTTCTTGAGCTCTCAGCCGGCAGAGATGACACCGCAC-5'.

[0087] SOD-F: 3'-CCGTCCGGGAAGACCACGGTGGCGGGCA-5'.

[0088] Constructing recombinant plasmids: Constructing the recombination system (30 μL): Linear template: 0.02 × fragment length (bp) = required fragment mass (ng), calculate the volume required based on the PCR product concentration.

[0089] SOD with homologous arms: 0.02 × fragment length (bp) = required fragment mass (ng), calculate the volume dosage based on the PCR product concentration.

[0090] Add 2×CE recombinase, bring the volume to 30 μL with double-distilled water, and run the program at 50℃ for 5 min to obtain the recombinant plasmid.

[0091] Protein expression and purification: The reconstructed nanocompartment protein particles were transformed into Escherichia coli for prokaryotic expression to obtain engineered E. coli. The engineered E. coli were then subjected to high pressure (1000 Pa) with buffer 1 (20 mM MOPS, 100 mM NaCl, pH 7.4) and centrifuged (18000 rpm, 40 min) to collect the supernatant.

[0092] The supernatant was purified by Flag-labeled affinity chromatography: the supernatant was loaded onto a chromatographic column packed with Anti-Flag packing material, the flag peptide was diluted with buffer1 to a concentration of 0.15 mg / ml, and eluted. The eluent was added to a concentration tube with a molecular weight cutoff of 100 kDa and centrifuged (3500 rpm) to less than 1 ml to obtain the crude extract of modified E. coli nanocompartment protein.

[0093] Equilibrate the Superose 6 Increase 10 / 300 size-exclusion column with one column volume (25 ml) of buffer 1. Load the crude extract onto the Superose 6 Increase 10 / 300 size-exclusion column and elute with 25 ml of buffer 1. The results are as follows. Figure 12 .Depend on Figure 12 It can be seen that the protein exhibits a characteristic peak between 10-14 ml.

[0094] Protein identification: Samples were collected at the characteristic peak positions to obtain a modified *E. coli* nanocompartment protein solution. This solution was analyzed by SDS-PAGE (the solution was treated with 5x loading buffer and the protein was denatured in a 100°C metal bath for 1 hour). The results are as follows: Figure 13 .Depend on Figure 13It can be seen that CFP-29 is located at approximately 29 kDa, while there is no corresponding SOD band at approximately 20 kDa. This indicates that the obtained sample's nanocompartment protein does not encapsulate SOD protein, proving that the DyP-linker, the N-terminal targeting peptide of DyP-type peroxidase, is a key sequence in the protein encapsulation process.

[0095] Example 3 The Enc (SOD) and SOD prepared in Example 1 were tested for their ability to reduce cellular oxidative stress and their stability.

[0096] The steps for testing the ability to reduce cellular oxidative stress are as follows: Add 100 μl of cell suspension containing 8000 293T cells to each well of a 96-well plate (3 columns in total), and culture in a cell culture incubator (37°C, 5% CO2) until the cell density is approximately 80%.

[0097] After removing the original culture medium and washing away the residual culture medium with PBS, DEME medium, DEME medium containing 5 mM hydrogen peroxide, and DEME medium containing 5 mM hydrogen peroxide and 1 mg / ml Enc (SOD) or 1 mg / ml SOD were added to each column of cells, and the cells were cultured in a cell culture incubator (37℃, 5% carbon dioxide) for 10 h.

[0098] Add 10 μl of CCK8 solution to each well, incubate in a cell culture incubator for 1 h, and measure the absorbance (at 450 nm wavelength) using a microplate reader. like Figures 14-15 As shown, cell growth was significantly inhibited by DEME medium containing 5 mM hydrogen peroxide, while cell growth was better by DEME medium containing 5 mM hydrogen peroxide and 1 mg / ml protein. Both SOD and Enc (SOD) showed the ability to reduce cellular oxidative stress, and there was no significant difference between them.

[0099] SOD stability testing includes enzyme digestion stability testing, temperature stability testing, and pH stability testing. The testing methods are as follows.

[0100] Enzyme digestion stability test: 1 mg / ml protein solution (Enc(SOD) or SOD) was mixed with 0.25% trypsin at a 1:1 ratio and incubated at 37°C for 1 hour, followed by SDS-PAGE analysis (15% gel concentration). Results are as follows: Figure 16 and Figure 17As shown, the band depth of the SOD group reacting with trypsin was significantly reduced, and there were even no obvious bands, proving that SOD was cleaved by trypsin. On the other hand, the band depth of the Enc (SOD) group reacting with trypsin was the same as that of the negative control group, and the corresponding SOD band could be clearly seen, proving that Enc can protect SOD from trypsin cleavage and enhance the enzyme resistance stability of SOD.

[0101] Temperature stability test: Enc(SOD) was diluted to 0.2 mg / ml with buffer 1 and incubated at 25, 42, 65, and 100℃ for 1 h. Then, the corresponding volume of 5×SDS loading was added to the protein solutions incubated at each of the four temperatures, and SDS-PAGE (15%) was performed. Results are as follows: Figure 18 As shown, Enc maintains structural stability within the temperature range of 25-37 degrees Celsius, with only a small amount of depolymerization; it also maintains partial structural stability at 65 degrees Celsius.

[0102] pH stability test: Buffer 1 was adjusted to pH 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. Enc (SOD) was diluted to 0.2 mg / ml using Buffer 1 at 11 different pH values. After incubation at 4°C for 1 h, the corresponding volumes of 5×SDS loading solution were added to the protein solutions at the 11 different pH values, and SDS-PAGE (15%) was performed. Results are as follows... Figure 19 As shown, Enc maintains structural stability within the pH range of 3-12, with only a small amount of depolymerization.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A complex protein, characterized in that, It includes a nanocompartment protein and a cargo protein loaded within the nanocompartment protein, wherein the nanocompartment protein loads the cargo protein via a targeting peptide, the amino acid sequence of which is shown in Seq. ID No.

1.

2. The composite protein according to claim 1, characterized in that, The nanocompartment protein includes the CFP-29 protein, the amino acid sequence of which is shown in Seq. ID No.

2.

3. The composite protein according to claim 1, characterized in that, The cargo proteins include one or more of the following: tyrosine recombinase Flp, peroxidase Dyp, superoxide dismutase SOD, green fluorescent protein GFP, folic acid biosynthesis protein FolB, bacterial ferritin Bfr, ​​ferricredoxin Fd, iron mineralization active encapsulation protein-associated Firmicutes protein IMEF, nitrite reductase domain and hydroxylamine oxidoreductase domain fusion protein NIR-HAO, earthworm-like hemoglobin, erythroglobin, and biphenyl dehydrogenase protein.

4. The composite protein according to claim 3, characterized in that, The cargo proteins include superoxide dismutase (SOD) and / or green fluorescent protein (GFP).

5. The composite protein according to claim 1, characterized in that, The diameter of the nanocompartment protein is 24 nm to 42 nm.

6. The composite protein according to claim 1, characterized in that, The complex protein satisfies at least one of the following: (1) The complex protein has a greater resistance to enzymatic digestion than the cargo protein; (2) The stability of the composite protein at 25℃~65℃ is greater than that of the cargo protein; (3) The stability of the composite protein in an environment with a pH of 3 to 12 is higher than that of the cargo protein.

7. A recombinant plasmid for expressing the complex protein according to any one of claims 1 to 6, characterized in that, The recombinant plasmid includes a sequence as shown in Seq.IDNo.

3.

8. The recombinant plasmid according to claim 7, characterized in that, The recombinant plasmid includes a sequence as shown in Seq.IDNo.

4.

9. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli includes the recombinant plasmid as described in any one of claims 7 to 8 and / or secretes the complex protein as described in any one of claims 1 to 6.

10. The use of a composite protein according to any one of claims 1 to 6 in enzyme catalysis, cell engineering, imaging detection, or fluorescent labeling.

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