A recombinant protein based on HIC1 functional domain optimization and its preparation method and application

Abstract

The present invention provides a recombinant protein optimized based on the functional domain of HIC1, as well as its preparation method and application. The recombinant protein comprises the following structures, from N-terminus to C-terminus, in order: a cell-penetrating peptide, a BTB / POZ domain, a rigid linker, a zinc finger structure, a flexible linker, and a nuclear localization signal. The amino acid sequence of the recombinant protein is shown in SEQ ID NO. 4, and the nucleotide sequence encoding the amino acids is shown in SEQ ID NO. 3. The present invention replaces the coding sequences of the BTB / POZ domain and the zinc finger domain with codons preferred by Escherichia coli, reducing the GC content from 67% to 52%. The resulting recombinant protein can be efficiently expressed in Escherichia coli. Simultaneously, the cell-penetrating peptide and the nuclear localization signal are integrated into the same recombinant protein, achieving "transfection-free" tumor suppressor function and providing a breakthrough solution for targeted tumor therapy.

Description

Technical Field

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

[0002] HIC1 ( Hypermethylated in Cancer 1) It is a key tumor suppressor gene, encoding proteins that regulate biological processes such as gene expression, cell cycle, apoptosis, and DNA damage repair through multiple mechanisms. HIC1 is located on human chromosome 17p13.3. Its gene expression exhibits dysmethylation in various tumors, leading it to be considered a potential cancer suppressor. Studies have shown that HIC1 exerts its tumor suppressor effects primarily through the following mechanisms: Recruitment of the transcriptional repressor complex: The BTB / POZ domain (47-110 aa) mediates binding to co-repressors such as HDACs and BCOR, silencing the expression of oncogenes such as SIRT1 and Cyclin D1. DNA targeting: The zinc finger domain (439-613 aa) specifically recognizes the promoter regions of target genes, such as the pro-apoptotic gene BAX. Nuclear-dependent function: HIC1 requires nuclear localization to exert its tumor suppressor activity, but the specific molecular mechanisms of its nuclear import remain undetermined.

[0003] Despite the clear potential of HIC1 in cancer therapy, the clinical application of its full-length protein (714 aa, approximately 65 kDa) is limited by two major bottlenecks: expression difficulties and low delivery efficiency. The following technical deficiencies and unresolved issues exist. Prokaryotic expression barriers: The yield of the full-length HIC1 protein is extremely low when expressed in Escherichia coli. The native sequence contains numerous codons with low frequency in E. coli (e.g., CGG for Arg, CCC for Pro, and GGA for Gly). Furthermore, the hydrophobic core of the BTB / POZ domain and the cysteine ​​residues in the zinc finger domain are prone to inclusion body formation, significantly reducing protein expression efficiency. Eukaryotic expression is costly: While mammalian or yeast systems can express soluble proteins, the processes are complex and time-consuming, making them difficult to meet industrial needs. The development of functional fragments is limited: Existing research focuses on expressing either the BTB / POZ or zinc finger domains alone, but the synergistic effect between the two is lacking. Due to its large molecular weight, native HIC1 has difficulty in nuclear entry, resulting in low efficiency of its tumor suppressor function. Furthermore, the HIC1 gene has not yet been used in breast cancer therapy.

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

[0005] In light of this, the present invention provides a recombinant protein optimized based on the functional domains of HIC1. Through multi-dimensional engineering, this approach effectively addresses the imbalance between expression efficiency, functional integrity, and delivery efficiency of HIC1 proteins in prokaryotes. This results in a soluble recombinant protein that can be efficiently and cost-effectively expressed in prokaryotes, integrating the synergistic effects of the BTB / POZ and zinc finger domains, and enabling precise targeted functional production.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] One of the objectives of the present invention is to provide a recombinant protein based on the optimization of the HIC1 functional domain, wherein the recombinant protein comprises 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.

[0008] In some specific embodiments, preferably, the cell-penetrating peptide sequence is: YGRKKRRQRRR;

[0009] The rigid linker sequence is: EAAAAK;

[0010] The flexible linker sequence is: GGGGSGGGG;

[0011] The nuclear localization signal sequence is: PKKKRKV.

[0012] Furthermore, the amino acid sequence of the recombinant protein is shown in SEQ ID NO.4.

[0013] The second object of the present invention is to provide a nucleotide sequence encoding the above-mentioned recombinant protein, wherein the nucleotide sequence is shown in SEQ ID NO.3.

[0014] The third object of the present invention is to provide an expression vector containing the above-mentioned nucleotide sequence.

[0015] In some specific embodiments, preferably, the vector is a pet28a vector.

[0016] A fourth object of the present invention is to provide an engineered bacterium containing the above-mentioned expression vector.

[0017] In some specific embodiments, preferably, the engineered bacteria is Escherichia coli Escherichia coli CICC ® 23796.

[0018] A fourth object of the present invention is to provide a method for preparing the above-mentioned recombinant protein, comprising the following steps:

[0019] 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. The expression was induced by IPTG, the bacteria were lysed, and the lysate was purified by Ni-NTA chromatography to obtain a soluble recombinant protein.

[0020] A fifth object of the present invention is to provide the use of the recombinant protein in the preparation of drugs for treating breast cancer.

[0021] A sixth object of the present invention is to provide a composition comprising the above-mentioned recombinant protein.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) Through multi-dimensional engineering transformation (codon optimization, domain truncation, functional tag fusion and stability design), the present invention achieves efficient expression and direct application of the HIC1 functional domain in prokaryotic cells, providing a breakthrough solution for targeted tumor therapy.

[0024] (2) Without changing the amino acid sequence, the present invention replaces the coding sequences of BTB / POZ (47-110aa) and zinc finger domain (439-613aa) with codons preferred by Escherichia coli, reducing the GC content from 67% to 52%, significantly improving the expression efficiency; further, the TAT transmembrane peptide (cell penetration) and SV40 nuclear localization signal (NLS) are integrated into the same recombinant protein to achieve a "transfection-free" tumor suppressor function.

[0025] (3) Compared with the unoptimized full-length protein, the recombinant protein provided by this application is expressed in the cell nucleus, achieving targeting. In the experiment on the breast cancer cell line MDA-MB-231 cells, the recombinant protein provided by this application caused its activity to drop to 51%, while the unoptimized full-length protein was only 72%, showing obvious advantages in tumor targeted therapy function. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the complete plasmid map before optimization of the recombinant protein provided in Example 1 of the present invention;

[0027] Figure 2 This is the complete plasmid map after optimization of the recombinant protein provided in Example 1 of the present invention;

[0028] Figure 3 This is a diagram showing the results of Western blot detection of purified protein using a His tag provided in Example 2 of the present invention;

[0029] Figure 4This is a comparison of the nuclear localization of the full-length HIC1 protein and the recombinant protein provided in Example 3 of the present invention;

[0030] Figure 5 This is a graph showing the effects of the full-length protein and the recombinant protein provided in Example 3 of the present invention on the viability of MDA-MB-231 cells. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0032] Example 1

[0033] This example provides a design and sequence optimization process for a recombinant protein based on HIC1 functional domain optimization, as follows:

[0034] 1.1 HIC1 functional domain design and codon optimization

[0035] Based on the sequence of the human HIC1 protein (UniProt ID: Q14526), ​​the core functional domains (BTB / POZ domain: 47-110 aa; zinc finger domain: 439-613 aa) were truncated, and a nuclear import signal (NLS) and a transmembrane peptide (TAT) were added to obtain the base sequence (SEQ ID NO. 1) and amino acid sequence (SEQ ID NO. 2) of the recombinant protein. The order of recombinant protein construction was: N-terminus → transmembrane peptide (TAT) → BTB / POZ domain → rigid linker → zinc finger structure → flexible linker → nuclear localization signal (NLS: SV40) → C-terminus.

[0036] SEQ ID NO.1 is as follows: ATGTACGGTCGCAAAAAACGTCGCCAGCGCCGTCGCTGCGACGTGATCATCGTGGTGCAGAACGCCCTCTTCCGCGCGCACAAGAACGTGCTGGCGGCCAGCAGCGCCTACCTCAAGTCCCTGGTGGTGCATGACAACCTGCTCAACCTGGACCATGACATGGTGAGCCCGGCCGTGTTCCGCCTGGTGCTGGACTTCATCTACACCGGCCGCCTGGCTGACGGCGCAGAAGCCGCCGCCGCCAAGTGCATTCCGTGCGGCAAGGGCTTCCCCAGCTCTGAGCAGCTGAACGCGCACGTGGAGGCTCACGTGGAGGAGGAGGAAGCGCTGTACGGCAGGGCCGAGGCGGCCGAAGTGGCCGCTGGGGCCGCCGGCCTAGGGCCCCCTTTTGGAGGCGGCGGGGACAAGGTCGCCGGGGCTCCGGGTGGCCTGGGAGAGCTGCTGCGGCCCTACCGCTGCGCGTCGTGCGACAAGAGCTACAAGGACCCGGCCACGCTGCGGCAGCACGAGAAGACGCACTGGCTGACCCGGCCCTACCCATGCACCATCTGCGGGAAGAAGTTCACGCAGCGTGGGACCATGACGCGCCACATGCGCAGCCACCTGGGCCTCAAGCCCTTCGCGTGCGACGCGTGCGGCATGCGGTTCACGCGCCAGTACCGCCTCACGGAGCACATGCGCATCCACTCGGGCGAGAAGCCCTACGAGTGCCAGGTGTGCGGCGGCAAGTTCGCACAGCAACGCAACCTCATCAGCCACATGAAGATGCACGGTGGTGGTGGTTCGGGTGGTGGTGGTGGTCCGAAGAAGAAGCGCAAGGTG。

[0037] SEQ ID NO.2 is as follows: MYGRKKRRQRRRCDVIIVVQNALFRAHKNVLAASSAYLKSLVVHDNLLNLDHDMVSPAVFRLVLDFIYTGRLADGAEAAAAKCIPCGKGFPSSEQLNAHVEAHVEEEEALYGRAEAAEVAAGAAGLGPPFGGG GDKVAGAPGGLGELLRPYRCASCDKSYKDPATLRQHEKTHWLTRPYPCTICGKKFTQRGTMTRHMRSHLGLKPFACDACGMRFTRQYRLTEHMRIHSGEKPYECQVCGGKFAQQRNLISHMKMHGGGGSGGGGGPKKKRKV.

[0038] In order to better express the recombinant protein in Escherichia coli, the entire sequence was optimized for Escherichia coli preferred codons. The optimized sequence is shown in SEQ ID NO.3. The amino acid expressed by the optimized sequence (SEQ ID NO.4) remains consistent with the amino acid sequence before optimization (SEQ ID NO.2).

[0039] SEQ ID NO. 3 is as follows: ATGTACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTTGCGACGTTATCATCGTTGTTCAGAACGCTCTGTTCCGTGCTCACAAAAACGTTCTGGCTGCTTCTTCTGCTTACCTGAAATCTCTGGTTGTTCACGACAACCTGCTGAACCTGGACCACGACATGGTTTCTCCGGCTGTTTTCCGTCTGGTTCTGGACTTCATCTACACCGGTCGTCTGGCTGACGGTGCTGAAGCTGCTGCTGCTAAATGCATCCCGTGCGGTAAAGGTTTCCCGTCTTCTGAACAGCTGAACGCTCACGTTGAAGCTCACGTTGAAGAAGAAGAAGCTCTGTACGGTCGTGCTGAAGCTGCTGAAGTTGCTGCTGGTGCTGCTGGTCTGGGTCCGCCGTTCGGTGGTGGTGGTGACAAAGTTGCTGGTGCTCCGGGTGGTCTGGGTGAACTGCTGCGTCCGTACCGTTGCGCTTCTTGCGACAAATCTTACAAAGACCCGGCTACCCTGCGTCAGCACGAAAAAACCCACTGGCTGACCCGTCCGTACCCGTGCACCATCTGCGGTAAAAAATTCACCCAGCGTGGTACCATGACCCGTCACATGCGTTCTCACCTGGGTCTGAAACCGTTCGCTTGCGACGCTTGCGGTATGCGTTTCACCCGTCAGTACCGTCTGACCGAACACATGCGTATCCACTCTGGTGAAAAACCGTACGAATGCCAGGTTTGCGGTGGTAAATTCGCTCAGCAGCGTAACCTGATCTCTCACATGAAAATGCACGGTGGTGGTGGTTCTGGTGGTGGTGGTGGTCCGAAAAAGAAACGTAAAGTT。

[0040] Comparing the sequences before and after optimization, protein expression efficiency was significantly improved after optimization: the codon adaptation index (CAI) increased from 0.34 to 1.0 (codons were optimized and usage index was calculated using the Codon Adaptation Tool), rare codons were completely eliminated, translation rate and accuracy were enhanced, and solubility was improved; GC content was homogenized to reduce mRNA secondary structure, lower the risk of translation stalling, increase the proportion of soluble protein, and enhance stability; ribosome queuing caused by rare codons was avoided, reducing protein truncation or misfolding.

[0041] The recombinant plasmid was designed based on the fragment of the recombinant protein. The base sequence of the recombinant protein plasmid before optimization is shown in SEQ ID NO.5. The map of the recombinant protein plasmid before optimization is shown in Figure 1 The optimized recombinant protein base sequence is shown in SEQ ID NO.6, and the optimized recombinant protein base sequence is shown in Figure 2 It should be noted that 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.

[0042] Example 2

[0043] This example constructs a prokaryotic expression vector based on the optimized sequence obtained in Example 1, as follows:

[0044] 2.1 Vector construction

[0045] (1) Restriction site design and sequence synthesis

[0046] According to the optimized sequence (SEQ ID NO.3), EcoRI and SalI The gene fragment, the synthesized base fragment is shown as SEQ ID NO.7.

[0047] SEQ ID NO. 7 is as follows: TCGCGGATCCGAATTCGATGTACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTTGCGACGTTATCATCGTTGTTCAGAACGCTCTGTTCCGTGCTCACAAAAACGTTCTGGCTGCTTCTTCTGCTTACCTGAAATCTCTGGTTGTTCACGACAACCTGCTGAACCTGGACCACGACATGGTTTCTCCGGCTGTTTTCCGTCTGGTTCTGGACTTCATCTACACCGGTCGTCTGGCTGACGGTGCTGAAGCTGCTGCTGCTAAATGCATCCCGTGCGGTAAAGGTTTCCCGTCTTCTGAACAGCTGAACGCTCACGTTGAAGCTCACGTTGAAGAAGAAGAAGCTCTGTACGGTCGTGCTGAAGCTGCTGAAGTTGCTGCTGGTGCTGCTGGTCTGGGTCCGCCGTTCGGTGGTGGTGGTGACAAAGTTGCTGGTGCTCCGGGTGGTCTGGGTGAACTGCTGCGTCCGTACCGTTGCGCTTCTTGCGACAAATCTTACAAAGACCCGGCTACCCTGCGTCAGCACGAAAAAACCCACTGGCTGACCCGTCCGTACCCGTGCACCATCTGCGGTAAAAAATTCACCCAGCGTGGTACCATGACCCGTCACATGCGTTCTCACCTGGGTCTGAAACCGTTCGCTTGCGACGCTTGCGGTATGCGTTTCACCCGTCAGTACCGTCTGACCGAACACATGCGTATCCACTCTGGTGAAAAACCGTACGAATGCCAGGTTTGCGGTGGTAAATTCGCTCAGCAGCGTAACCTGATCTCTCACATGAAAATGCACGGTGGTGGTGGTTCTGGTGGTGGTGGTGGTCCGAAAAAAAAACGTAAAGTTGTCGACAAGCTTGCGGC。

[0048] (2) Double digestion of the vector and the inserted fragment

[0049] The synthesized gene fragment (SEQ ID NO. 7) and the pET28a(+) vector were respectively digested with EcoRI and SalIDouble enzyme digestion was performed, and the digestion products were purified and recovered by 1% agarose gel electrophoresis.

[0050] (3) Connection and transformation

[0051] Use T4 DNA ligase to ligate the target gene to the linearized vector, then transform the ligation product into Escherichia coli DH5α competent cells, spread on LB plates containing kanamycin, and culture at 37°C for 12-16 hours.

[0052] (4) Positive clone screening

[0053] Sequencing verification: Single colonies were picked, amplified, and plasmids were extracted and sent to Sangon Biotech Co., Ltd. for Sanger sequencing to confirm that the obtained plasmid sequence was completely consistent with SEQ ID NO.6.

[0054] 2.2 Recombinant protein expression and purification

[0055] (1) Inducible expression

[0056] The correct recombinant plasmid was transformed into Escherichia coli ( Escherichia coli CICC ® 23796), induce according to the following process:

[0057] 1) Inoculate a single colony into LB liquid medium containing kanamycin and culture at 37°C with shaking until the OD 600 =0.6;

[0058] 2) Add IPTG inducer at a final concentration of 0.5 mM and induce at 18°C ​​for 16 hours to promote soluble expression;

[0059] 3) Collect the cells by centrifugation at 4°C (13,000 × g, 10 min).

[0060] (2) Protein purification

[0061] 1) Resuspend the cells in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0).

[0062] 2) Ultrasonic disruption in an ice bath (200W, 3 seconds on / 5 seconds off, for a total of 15-20 minutes), and centrifugation at 4°C, 13,000 × g, for 10 minutes to collect the supernatant;

[0063] 3) Incubate the supernatant with Ni-NTA magnetic beads at 4°C for 1 hour. Discard the supernatant after magnetic separation. Elute the beads with buffers containing 20mM and 50mM imidazole to remove nonspecific binding. Finally, elute the beads with buffer containing 250mM imidazole to collect the target protein.

[0064] 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.

[0065] 2.3 Recombinant protein identification

[0066] Western blot verification: The purified protein obtained in the previous step was subjected to Western blot detection, and the specificity of the protein was detected using an anti-His tag monoclonal antibody. The results are shown in Figure 3 The results showed that: using Escherichia coli ( Escherichia coli CICC ® 23796) expressed recombinant protein. Before IPTG induction, no specific protein was produced. After IPTG induction, the optimized recombinant protein was expressed, and a specific protein was detected by His monoclonal antibody. After IPTG induction, the optimized recombinant protein was expressed, and the 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.

[0067] Example 3

[0068] This example compares the functions of the recombinant protein obtained in Example 2 and the full-length HIC1 protein, as follows:

[0069] Obtaining the full-length HIC1 protein

[0070] Based on the sequence of the HIC1 protein (UniProt ID: Q14526), ​​it was conjugated to a cell-penetrating peptide and a nuclear localization sequence, and a recombinant plasmid was constructed using pET28a(+) as the vector. (The plasmid sequence is shown in SEQ ID NO. 8, which contains the following structure from N-terminus to C-terminus: cell-penetrating peptide, full-length HIC1 sequence, flexible linker, and nuclear localization signal. It should be noted that SEQ ID NO. 8 is too long to be shown in the specification, but is shown in the sequence listing.) The purified protein, i.e., the full-length HIC1 protein, was then obtained using the same steps as in Example 2.

[0071] 3.2 Comparison of nuclear localization ability between recombinant protein and full-length HIC1 protein:

[0072] The breast cancer cell line MDA-MB-231 was cultured. One group of MDA-MB-231 cells was incubated with 50 μg / mL of the aforementioned full-length HIC1 protein; another group of cells was incubated with 50 μg / mL of the recombinant protein. After 24 hours, His-tag immunofluorescence was used to visualize nuclear localization, combined with DAPI nuclear staining.

[0073] The results showed that the full-length HIC1 protein was mainly expressed in the nucleus, but also partially localized in the cytoplasm; while the optimized recombinant proteins were all expressed in the nucleus (see Figure 4 ).

[0074] 3.3 CCK8 assay to detect cell viability

[0075] Breast cancer cell line MDA-MB-231 cells were cultured, and the experimental groups were set up as follows:

[0076] Blank group: cells were treated with PBS only;

[0077] Negative control group: cells were incubated with 50 μg / mL purified membrane-penetrating peptide;

[0078] Full-length protein group: cells were incubated with 50 μg / mL HIC1 full-length purified protein;

[0079] Recombinant protein group: cells were incubated with 50 μg / mL recombinant protein.

[0080] Each group was incubated for 72 h, and then the cell viability was detected using the CCK8 kit.

[0081] The results showed that: incubation of MDA-MB-231 cells with cell-penetrating peptides did not affect their cell viability; incubation of MDA-MB-231 cells with HIC1 full-length protein caused their viability to decrease to 72% (p≤0.05); incubation of MDA-MB-231 cells with recombinant protein caused their viability to decrease to 51% (p≤0.05) (results see Figure 5 ).

[0082] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant protein based on the optimization of HIC1 functional domain, characterized in that: The recombinant protein comprises the following structures from N-terminus to C-terminus: a membrane-penetrating peptide, a BTB / POZ domain, a rigid linker, a zinc finger structure, a flexible linker, and a nuclear localization signal. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

4.

2. The recombinant protein according to claim 1, characterized in that The cell-penetrating peptide sequence is: YGRKKRRQRRR; The rigid linker sequence is: EAAAAK; The flexible linker sequence is: GGGGSGGGG; The nuclear localization signal sequence is: PKKKRKV.

3. The nucleotide sequence encoding the recombinant protein according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

3.

4. An expression vector containing the nucleotide sequence of claim 3.

5. An engineered bacterium containing the expression vector according to claim 4.

6. The engineered bacteria according to claim 5, characterized in that The engineered bacteria is Escherichia coli Escherichia coli CICC ® 23796.

7. A method for preparing the recombinant protein according to claim 1 or 2, characterized in that: The following steps are involved: The nucleotide sequence shown in SEQ ID NO.3 is inserted into a vector to obtain an expression vector, and the vector is transformed into the Escherichia coli according to claim 6. The expression is induced by IPTG, the bacteria are lysed, and the lysate is purified by Ni-NTA chromatography to obtain a soluble recombinant protein.

8. Use of the recombinant protein according to claim 1 or 2 in the preparation of a drug for treating breast cancer.

9. A composition, characterized in that The composition comprises the recombinant protein according to claim 1 or 2.

Images