Recombinant protein based on HIC1 functional structural domain optimization and preparation method and application thereof

Abstract

The invention provides a recombinant protein based on HIC1 functional structural domain optimization and a preparation method and application thereof. The recombinant protein sequentially comprises the following structures from the N end to the C end: cell-penetrating peptide, a BTB / POZ structural domain, a rigid connexon, a zinc finger structure, a flexible connexon and a nuclear localization signal. The amino acid sequence of the recombinant protein is as shown in SEQ ID NO.4, and the nucleotide sequence for coding the amino acid is as shown in SEQ ID NO.3. Coding sequences of a BTB / POZ structural domain and a zinc finger structural domain are replaced by escherichia coli preference codons, the GC content is reduced from 67% to 52%, the provided recombinant protein can be efficiently expressed in escherichia coli, cell-penetrating peptide and a nuclear localization signal are integrated into the same recombinant protein, the cancer suppression function without transfection is achieved, and the recombinant protein has the advantages of being simple in structure, low in cost and high in efficiency. And a breakthrough solution is provided for tumor targeted therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of recombinant proteins, and particularly relates to a recombinant protein optimized based on the functional domain of HIC1, and a preparation method and application thereof. Background Art

[0002] HIC1 ( Hypermethylated in Cancer 1) is an important tumor suppressor gene, and the encoded protein regulates biological processes such as gene expression, cell cycle, apoptosis, and DNA damage repair through multiple mechanisms. HIC1 is located in the human chromosome region 17p13.3, and its gene expression shows methylation dysregulation in various tumors, so it is considered a potential cancer suppressor. Research shows that HIC1 mainly exerts its anti-cancer effect through the following mechanisms. Transcriptional repression complex recruitment: The BTB / POZ domain (47 - 110aa) mediates binding to co-repressors such as HDACs and BCOR, silencing the expression of oncogenes (such as SIRT1 and Cyclin D1). DNA-targeted binding: The zinc finger domain (439 - 613aa) specifically recognizes the promoter region of target genes, such as the pro-apoptotic gene BAX. Nuclear-dependent function: HIC1 needs to be localized in the nucleus to exert its anti-cancer activity, but the specific molecular mechanism of its nuclear import has not been resolved yet.

[0003] Although HIC1 has clear potential in tumor treatment, the clinical application of its full-length protein (714aa, about 65 kDa) is limited by two major bottlenecks: difficult expression and low delivery efficiency. Currently, there are the following technical defects and unsolved problems. Prokaryotic expression obstacle: When the full-length HIC1 protein is expressed in Escherichia coli, the yield is extremely low. The natural sequence contains a large number of Escherichia coli low-frequency codons (such as CGG for Arg, CCC for Pro, and GGA for Gly), and the hydrophobic core of the BTB / POZ domain and the cysteine of the zinc finger domain are prone to inclusion body formation, significantly reducing the protein expression efficiency. High cost of eukaryotic expression: Although soluble proteins can be expressed in mammalian or yeast systems, the process is complex, the cycle is long, and it is difficult to meet the industrial demand. Insufficient development of functional fragments: Existing research focuses on the individual expression of the BTB / POZ or zinc finger domain, but the synergistic effect between the two is lacking; due to its large molecular weight, natural HIC1 has difficulty entering the nucleus, and its anti-cancer function efficiency is low. In addition, there is no application of the HIC1 gene in breast cancer treatment yet.

[0004] Based on this, there is an urgent need to provide a soluble recombinant protein that can be efficiently and low-cost expressed in prokaryotes, simultaneously achieve the synergistic effect of the BTB / POZ domain and the zinc finger domain, and can target breast cancer treatment. Summary of the Invention

[0005] In view of this, the present invention provides a recombinant protein optimized based on the functional domain of HIC1. Through multi-dimensional engineering transformation, the imbalance problem of the expression efficiency, functional integrity and delivery efficiency of HIC1 protein in the prokaryotic system is finally effectively solved. A soluble recombinant protein that can be highly efficiently and low-cost expressed by prokaryotes, combines the synergistic effects of the BTB / POZ domain and the zinc finger domain, and can accurately target and generate functions is obtained.

[0006] To achieve the above object, the present invention adopts the following technical solutions: One of the objects of the present invention is to provide a recombinant protein optimized based on the functional domain of HIC1, and the recombinant protein sequentially includes the following structures from the N-terminus to the C-terminus: a transmembrane peptide, a BTB / POZ domain, a rigid linker, a zinc finger structure, a flexible linker, and a nuclear localization signal.

[0007] In some specific embodiments, preferably, the sequence of the transmembrane peptide is: YGRKKRRQRRR; The sequence of the rigid linker is: EAAAK; The sequence of the flexible linker is: GGGGSGGGGS; The sequence of the nuclear localization signal is: PKKKRKV.

[0008] Furthermore, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO.4.

[0009] Another object of the present invention is to provide a nucleotide sequence encoding the above recombinant protein, and the nucleotide sequence is as shown in SEQ ID NO.3.

[0010] Another object of the present invention is to provide an expression vector containing the above nucleotide sequence.

[0011] In some specific embodiments, preferably, the vector is a pet28a vector.

[0012] Another object of the present invention is to provide an engineered bacterium containing the above expression vector.

[0013] In some specific embodiments, preferably, the engineered bacterium is Escherichia coli Escherichia coli CICC ® 23796.

[0014] Another object of the present invention is to provide a method for preparing the above recombinant protein, including the following steps: The nucleotide sequence shown in SEQ ID NO.3 was inserted into a vector to obtain an expression vector, which was then transformed into the above-mentioned Escherichia coli. IPTG was used to induce expression, the bacterial cells were lysed, and the lysate was purified by Ni-NTA chromatography to obtain the soluble recombinant protein.

[0015] A fifth object of the present invention is to provide the use of the recombinant protein in the preparation of a breast cancer treatment drug.

[0016] A sixth object of the present invention is to provide a composition comprising the above-mentioned recombinant protein.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Through multi-dimensional engineering transformation (codon optimization, domain truncation, functional tag fusion, and stability design), the present invention realizes the high-efficiency expression and direct application of the HIC1 functional domain in prokaryotic cells, providing a breakthrough solution for tumor targeted therapy.

[0018] (2) Without changing the amino acid sequence, the present invention replaces the coding sequences of BTB / POZ (47-110aa) and the zinc finger domain (439-613aa) with Escherichia coli-preferred codons, reducing the GC content from 67% to 52%, significantly improving the expression efficiency; further, the TAT cell-penetrating peptide and the SV40 nuclear localization signal (NLS) are integrated into the same recombinant protein to achieve the anti-cancer function of "no transfection required".

[0019] (3) Compared with the unoptimized full-length protein, the recombinant protein provided in the present application is expressed in the nucleus, achieving targeting; in the experiment on breast cancer cell line MDA-MB-231 cells, the recombinant protein provided in the present application causes its viability to drop to 51%, while the unoptimized full-length protein is only 72%, showing obvious advantages in the function of tumor targeted therapy. Description of the Drawings

[0020] Figure 1 It is the complete plasmid map of the recombinant protein before optimization provided in Example 1 of the present invention; Figure 2 It is the complete plasmid map of the recombinant protein after optimization provided in Example 1 of the present invention; Figure 3 It is the result diagram of detecting the purified protein by His tag in Western blot provided in Example 2 of the present invention; Figure 4 It is the nuclear localization comparison diagram of the HIC1 full-length protein and the recombinant protein provided in Example 3 of the present invention; Figure 5This is the diagram showing the effects of the full-length protein and recombinant protein provided in Example 3 of the present invention on the viability of MDA-MB-231 cells. Detailed implementation manners

[0021] The present invention will be further described in detail below in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagent consumables are commercially available products.

[0022] Example 1 This example provides a design and sequence optimization process of a recombinant protein based on the functional domain of HIC1, which is as follows: 1.1 Design and codon optimization of the HIC1 functional domain Based on the sequence of the human HIC1 protein (UniProt ID: Q14526), its core functional domains (BTB / POZ domain: 47-110aa; zinc finger domain: 439-613aa) are intercepted, and a nuclear import signal (NLS) and a transmembrane peptide (TAT) are added to obtain the base sequence (SEQ ID NO.1) and amino acid sequence (SEQ ID NO.2) of the recombinant protein. The construction order of the recombinant protein is N-terminal → transmembrane peptide (TAT) → BTB / POZ domain → rigid linker → zinc finger structure → flexible linker → nuclear localization signal (NLS: SV40) → C-terminal.

[0023] SEQ ID NO.1 is as follows: ATGTACGGTCGCAAAAAACGTCGCCAGCGCCGTCGCTGCGACGTGATCATCGTGGTGCAGAACGCCCTCTTCCGCGCGCACAAGAACGTGCTGGCGGCCAGCAGCGCCTACCTCAAGTCCCTGGTGGTGCATGACAACCTGCTCAACCTGGACCATGACATGGTGAGCCCGGCCGTGTTCCGCCTGGTGCTGGACTTCATCTACACCGGCCGCCTGGCTGACGGCGCAGAAGCCGCCGCCGCCAAGTGCATTCCGTGCGGCAAGGGCTTCCCCAGCTCTGAGCAGCTGAACGCGCACGTGGAGGCTCACGTGGAGGAGGAGGAAGCGCTGTACGGCAGGGCCGAGGCGGCCGAAGTGGCCGCTGGGGCCGCCGGCCTAGGGCCCCCTTTTGGAGGCGGCGGGGACAAGGTCGCCGGGGCTCCGGGTGGCCTGGGAGAGCTGCTGCGGCCCTACCGCTGCGCGTCGTGCGACAAGAGCTACAAGGACCCGGCCACGCTGCGGCAGCACGAGAAGACGCACTGGCTGACCCGGCCCTACCCATGCACCATCTGCGGGAAGAAGTTCACGCAGCGTGGGACCATGACGCGCCACATGCGCAGCCACCTGGGCCTCAAGCCCTTCGCGTGCGACGCGTGCGGCATGCGGTTCACGCGCCAGTACCGCCTCACGGAGCACATGCGCATCCACTCGGGCGAGAAGCCCTACGAGTGCCAGGTGTGCGGCGGCAAGTTCGCACAGCAACGCAACCTCATCAGCCACATGAAGATGCACGGTGGTGGTGGTTCGGGTGGTGGTGGTGGTCCGAAGAAGAAGCGCAAGGTG。

[0024] SEQ ID NO.2 is as follows: MYGRKKRRQRRRCDVIIVVQNALFRAHKNVLAASSAYLKSLVVHDNLLNLDHDMVSPAVFRLVLDFIYTGRLADGAEAAAAKCIPCGKGFPSSEQLNAHVEAHVEEEEALYGRAEAAEVAAGAAGLGPPFGGGGDKVAGAPGGLGELLRPYRCASCDKSYKDPATLRQHEKTHWLTRPYPCTICGKKFTQRGTMTRHMRSHLGLKPFACDACGMRFTRQYRLTEHMRIHSGEKPYECQVCGGKFAQQRNLISHMKMHGGGGSGGGGGPKKKRKV.

[0025] To enable better expression of the recombinant protein in Escherichia coli, the entire sequence was optimized for Escherichia coli-preferred codons. The optimized sequence is shown as SEQ ID NO.3, and the amino acids expressed by the optimized sequence (SEQ ID NO.4) are identical to the amino acid sequence before optimization (SEQ ID NO.2).

[0026] SEQ ID NO. 3 is as follows: ATGTACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTTGCGACGTTATCATCGTTGTTCAGAACGCTCTGTTCCGTGCTCACAAAAACGTTCTGGCTGCTTCTTCTGCTTACCTGAAATCTCTGGTTGTTCACGACAACCTGCTGAACCTGGACCACGACATGGTTTCTCCGGCTGTTTTCCGTCTGGTTCTGGACTTCATCTACACCGGTCGTCTGGCTGACGGTGCTGAAGCTGCTGCTGCTAAATGCATCCCGTGCGGTAAAGGTTTCCCGTCTTCTGAACAGCTGAACGCTCACGTTGAAGCTCACGTTGAAGAAGAAGAAGCTCTGTACGGTCGTGCTGAAGCTGCTGAAGTTGCTGCTGGTGCTGCTGGTCTGGGTCCGCCGTTCGGTGGTGGTGGTGACAAAGTTGCTGGTGCTCCGGGTGGTCTGGGTGAACTGCTGCGTCCGTACCGTTGCGCTTCTTGCGACAAATCTTACAAAGACCCGGCTACCCTGCGTCAGCACGAAAAAACCCACTGGCTGACCCGTCCGTACCCGTGCACCATCTGCGGTAAAAAATTCACCCAGCGTGGTACCATGACCCGTCACATGCGTTCTCACCTGGGTCTGAAACCGTTCGCTTGCGACGCTTGCGGTATGCGTTTCACCCGTCAGTACCGTCTGACCGAACACATGCGTATCCACTCTGGTGAAAAACCGTACGAATGCCAGGTTTGCGGTGGTAAATTCGCTCAGCAGCGTAACCTGATCTCTCACATGAAAATGCACGGTGGTGGTGGTTCTGGTGGTGGTGGTGGTCCGAAAAAGAAACGTAAAGTT。

[0027] Comparing the sequences before and after optimization, after optimization, the protein expression efficiency was significantly improved: the Codon Adaptation Index (CAI) increased from 0.34 to 1.0 (using the Codon Adaptation Tool to optimize codons and calculate the usage index), rare codons were completely eliminated, the translation rate and accuracy were enhanced, and the solubility was improved; the GC content was homogenized to reduce the mRNA secondary structure, reduce the risk of translation stalling, increase the proportion of soluble proteins, and enhance the stability; avoid the ribosome queuing phenomenon caused by rare codons, and reduce protein truncation or misfolding.

[0028] According to the fragments of the recombinant protein, a recombinant plasmid was designed. The base sequence of the recombinant protein plasmid before optimization is shown in SEQ ID NO.5, and the map of the recombinant protein plasmid before optimization is shown in Figure 1 ; the base sequence of the recombinant protein plasmid after optimization is shown in SEQ ID NO.6, and the map of the recombinant protein plasmid after optimization is shown in Figure 2 . It should be noted that the sequences SEQ ID NO.5 and SEQ ID NO.6 are relatively long and are not shown in the specification, but are shown in the sequence listing.

[0029] Example 2 In this example, a prokaryotic expression vector was constructed for the optimized sequence obtained in Example 1, as follows: 2.1 Vector construction (1) Restriction site design and sequence synthesis According to the optimized sequence (SEQ ID NO.3), a gene fragment containing EcoRI and SalI was synthesized, and the synthesized base fragment is shown in SEQ ID NO.7.

[0030] SEQ ID NO. 7 is as follows: TCGCGGATCCGAATTCGATGTACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTTGCGACGTTATCATCGTTGTTCAGAACGCTCTGTTCCGTGCTCACAAAAACGTTCTGGCTGCTTCTTCTGCTTACCTGAAATCTCTGGTTGTTCACGACAACCTGCTGAACCTGGACCACGACATGGTTTCTCCGGCTGTTTTCCGTCTGGTTCTGGACTTCATCTACACCGGTCGTCTGGCTGACGGTGCTGAAGCTGCTGCTGCTAAATGCATCCCGTGCGGTAAAGGTTTCCCGTCTTCTGAACAGCTGAACGCTCACGTTGAAGCTCACGTTGAAGAAGAAGAAGCTCTGTACGGTCGTGCTGAAGCTGCTGAAGTTGCTGCTGGTGCTGCTGGTCTGGGTCCGCCGTTCGGTGGTGGTGGTGACAAAGTTGCTGGTGCTCCGGGTGGTCTGGGTGAACTGCTGCGTCCGTACCGTTGCGCTTCTTGCGACAAATCTTACAAAGACCCGGCTACCCTGCGTCAGCACGAAAAAACCCACTGGCTGACCCGTCCGTACCCGTGCACCATCTGCGGTAAAAAATTCACCCAGCGTGGTACCATGACCCGTCACATGCGTTCTCACCTGGGTCTGAAACCGTTCGCTTGCGACGCTTGCGGTATGCGTTTCACCCGTCAGTACCGTCTGACCGAACACATGCGTATCCACTCTGGTGAAAAACCGTACGAATGCCAGGTTTGCGGTGGTAAATTCGCTCAGCAGCGTAACCTGATCTCTCACATGAAAATGCACGGTGGTGGTGGTTCTGGTGGTGGTGGTGGTCCGAAAAAAAAACGTAAAGTTGTCGACAAGCTTGCGGC。

[0031] (2)Double digestion of vector and inserted fragment The synthesized gene fragment (SEQ ID NO. 7) and the pET28a(+) vector were respectively digested with EcoRI and SalIPerform double digestion, and the digestion products are purified and recovered by 1% agarose gel electrophoresis.

[0032] (3)Ligation and transformation Use T4 DNA ligase to ligate the target gene with the linearized vector, and then transform the ligation product into competent Escherichia coli DH5α cells. Spread the cells on an LB plate containing kanamycin and culture at 37 °C for 12 - 16 hours.

[0033] (4)Screening of positive clones Sequencing verification: Pick a single colony, amplify and extract the plasmid, and send it to Sangon Biotech for Sanger sequencing to confirm that the obtained plasmid sequence is exactly the same as SEQ ID NO.6.

[0034] 2.2 Expression and purification of recombinant protein (1)Induced expression Transform the verified recombinant plasmid into Escherichia coli ( Escherichia coli CICC ® 23796), and induce according to the following procedure: 1) Inoculate a single colony into an LB liquid medium containing kanamycin and culture with shaking at 37 °C until OD 600 = 0.6; 2) Add IPTG inducer with a final concentration of 0.5 mM and induce at low temperature of 18 °C for 16 hours to promote soluble expression; 3) Centrifuge at 4 °C (13000×g, 10 minutes) to collect the bacterial cells.

[0035] (2)Protein purification 1) Resuspend the bacterial cells in lysis buffer (20 mM Tris - HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0); 2) Sonicate on ice (200 W, working for 3 seconds / interval of 5 seconds, a total of 15 - 20 minutes of cycling), centrifuge at 4 °C, 13000×g, 10 min to collect the supernatant; 3) Incubate the supernatant with Ni - NTA magnetic beads at 4 °C for 1 hour, separate with a magnetic stand and discard the supernatant. Wash the magnetic beads with buffers containing 20 mM and 50 mM imidazole in turn to remove non - specific binding, and elute the magnetic beads with a buffer containing 250 mM imidazole to collect the target protein; 4) Purify the target protein using a PD - 10 column, elute the target protein into PBS buffer (pH 7.4) and store at - 80 °C.

[0036] 2.3 Identification of recombinant protein Western blot verification: The purified protein obtained in the previous step was subjected to Western blot detection, and a monoclonal anti-His tag antibody was used to detect the specificity of the protein. The results are shown in Figure 3 . The results showed that when using Escherichia coli ( Escherichia coli CICC ® 23796) to express the recombinant protein, no specific protein was produced before IPTG induction; when using IPTG-induced expression of the optimized recombinant protein, a specific protein could be detected by the His monoclonal antibody; when using IPTG-induced expression of the optimized recombinant protein, a specific protein could be detected at the same molecular weight, and the protein expression level was increased by 3.8 times compared with that before optimization.

[0037] Example 3 In this example, a functional comparison was made between the recombinant protein obtained in Example 2 and the full-length HIC1 protein, as follows: 3.1 Obtaining the full-length HIC1 protein According to the sequence of the HIC1 protein (UniProt ID: Q14526), a transmembrane peptide was combined with a nuclear localization sequence, and pET28a(+) was used as the vector to construct a recombinant plasmid (the plasmid sequence is shown in SEQ ID NO.8, which contains the following structures in sequence from the N-terminus to the C-terminus: transmembrane peptide, full-length HIC1 sequence, flexible linker, nuclear localization signal. It should be noted that the sequence SEQ ID NO.8 is too long to be shown in the specification, but it has been shown in the sequence listing). Subsequently, the purified protein, that is, the full-length HIC1 protein, was obtained using the same steps as in Example 2.

[0038] 3.2 Comparison of the nuclear localization ability of the recombinant protein and the full-length HIC1 protein: The breast cancer cell line MDA-MB-231 cells were cultured. One group of MDA-MB-231 cells was incubated with 50 μg / mL of the above-mentioned full-length HIC1 protein; another group of cells was incubated with 50 μg / mL of the recombinant protein. After 24 h, the His tag was labeled by immunofluorescence technology and combined with DAPI for nuclear staining to observe the localization.

[0039] The results showed that the full-length HIC1 protein was mainly expressed in the nucleus, but some was also localized in the cytoplasm; while the optimized recombinant protein was expressed in the nucleus (the results are shown in Figure 4 ).

[0040] 3.3 Detection of cell viability by CCK8 assay The breast cancer cell line MDA-MB-231 cells were cultured. The experimental groups were set as follows: Blank group: Cells were only treated with PBS; Negative control group: Cells were incubated with 50 μg / mL purified cell-penetrating peptide; Full-length proteome group: Cells were incubated with 50 μg / mL full-length purified HIC1 protein; Recombinant protein group: Cells were incubated with 50 μg / mL recombinant protein.

[0041] Cells in each group were incubated for 72 hours, and then the cell viability was detected using a CCK8 kit.

[0042] The results showed that: Incubation of MDA-MB-231 cells with cell-penetrating peptide did not affect their cell viability; Incubation of MDA-MB-231 cells with full-length HIC1 protein led to a decrease in their viability to 72% (p≤0.05); Incubation of MDA-MB-231 cells with recombinant protein led to a decrease in their viability to 51% (p≤0.05) (the results are shown in Figure 5 ).

[0043] The above are only the preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A recombinant protein optimized based on the functional domain of HIC1, characterized in that, The recombinant protein sequentially contains the following structures from the N-terminus to the C-terminus: a transmembrane peptide, a BTB / POZ domain, a rigid linker, a zinc finger structure, a flexible linker, and a nuclear localization signal.

2. The recombinant protein according to claim 1, wherein The transmembrane peptide sequence is: YGRKKRRQRRR; The rigid linker sequence is: EAAAK; The flexible linker sequence is: GGGGGSGGGGS; The nuclear localization signal sequence is: PKKKRKV.

3. The recombinant protein according to claim 1 or 2, characterized in that, The amino acid sequence of the recombinant protein is as shown in SEQ ID NO.

4.

4. The nucleotide sequence encoding the recombinant protein according to claim 3, characterized in that, The nucleotide sequence is as shown in SEQ ID NO.

3.

5. An expression vector containing the nucleotide sequence recited in claim 4.

6. An engineered bacterium containing the expression vector recited in claim 5.

7. The engineered bacterium according to claim 6, wherein The engineered bacterium is Escherichia coli Escherichia coli CICC ® 23796.

8. A method for preparing the recombinant protein according to any one of claims 1-3, characterized in that, comprising the following steps: Insert the nucleotide sequence shown in SEQ ID NO.3 into a vector to obtain an expression vector, and transform it into the Escherichia coli recited in claim 7. Induce expression with IPTG, lyse the bacterial cells, and purify the lysate by Ni-NTA chromatography to obtain the soluble recombinant protein.

9. Use of the recombinant protein according to any one of claims 1-3 in the preparation of a medicament for treating breast cancer.

10. A composition, characterized in that, The composition contains the recombinant protein according to any one of claims 1-3.

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