A Cth TerA-containing peptide variant and its application in the biological preparation of tetrapeptide-7

Abstract

This invention discloses an intima-in-the-liquid peptide and its application in the biological preparation of tetrapeptide-7. The amino acid sequence of the Cth TerA intima-in-the-liquid peptide variant is shown in SEQ ID NO:3. In this invention, a highly efficient and stable recombinant expression vector for tetrapeptide-7 is prepared by combining the Cth TerA intima-in-the-liquid peptide variant with tetrapeptide-7 to form a fusion protein. After being introduced into a vector plasmid, an engineered bacterial strain is obtained. High-purity, unmodified tetrapeptide-7 can be obtained through induced expression. This method is simple, rapid, produces no toxic byproducts, and yields large quantities, making it suitable for large-scale industrial production of tetrapeptide-7. This results in high-quality tetrapeptide-7 with significant market value.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a Cth TerA inteptide variant and its application in the biological preparation of tetrapeptide-7. Background Technology

[0002] Palmitoyl tetrapeptide-7 can delay and inhibit the production of excessive interleukins, thereby suppressing some unnecessary or inappropriate inflammatory responses and glycosylation damage. In in vitro experiments, researchers found that palmitoyl tetrapeptide-7 induces a significant dose-dependent reduction in interleukin production; that is, the higher the dose of palmitoyl tetrapeptide-7 used, the greater the reduction in interleukins, with a maximum reduction of up to 40%.

[0003] Palmitoyl tetrapeptide-7 has extremely high application value. Related technologies indicate that UV radiation can promote the production of interleukins. When cells are exposed to UV radiation and treated with palmitoyl tetrapeptide-7, an 86% reduction in interleukins can be observed. Related technologies also show that palmitoyl tetrapeptide-7 can promote the growth of connective tissue and increase collagen production in the skin. Increased collagen allows the skin to achieve self-healing and regeneration. Furthermore, palmitoyl tetrapeptide-7 can accelerate the expression of granulocyte chemotactic protein-2 (GCP-2), promoting wound healing. Moreover, palmitoyl tetrapeptide-7 is a fragment of immunoglobulin IgG, which has many biological activities, especially immunomodulatory functions. Palmitoyl tetrapeptide-7 has extremely high application prospects in cosmetic products, especially anti-aging products. Related studies have found that skin cytokines, especially IL-6, participate in chronic inflammatory responses and play an important role in skin aging. During aging, the decrease in dehydroepiandrosterone (DHEA) and the increase in IL-6 show a strong correlation. Research has found that palmitoyl tetrapeptide-7 mimics DHEA in the skin, thereby controlling the levels of inflammatory cytokines in the circulatory system and reducing IL-6 levels associated with aging. This helps restore cytokine balance in the skin, resulting in improved skin health, reduced inflammation, and firmer, smoother, and more elastic skin. Furthermore, palmitoyl tetrapeptide-7 can act as a cell messenger to stimulate collagen fiber regeneration in the dermis and is believed to increase hyaluronic acid levels, thus helping to tighten the skin by drawing moisture to the epidermis. Palmitoyl tetrapeptide-7 is also commonly found in anti-aging serums, moisturizers, and certain cosmetic formulations. It has been shown to have a statistically significant effect on improving skin elasticity, reducing deep wrinkles, improving skin texture, and exhibiting a dose-responsive effect on collagen 1, fibronectin, and hyaluronic acid synthesis. Therefore, palmitoyl tetrapeptide-7 and its intermediate tetrapeptide-7 have significant market application value.

[0004] However, current industrial synthesis of tetrapeptide-7 primarily relies on chemical synthesis. This method is characterized by numerous byproducts, low yields, significant pollution, and high costs, making it difficult to meet the demands of a carbon-neutral era. While biosynthesis can effectively address the shortcomings of chemical synthesis, there is currently no efficient biosynthetic route for the complex structure of tetrapeptide-7. Moreover, current chemical synthesis of tetrapeptide-7 often generates numerous synthetic byproducts, some of which are highly cytotoxic. This results in substantial costs for the production and purification of tetrapeptide-7 and significant pollution, severely limiting its further development and application. Therefore, developing a greener, more economical, and efficient synthetic method is urgently needed for the industrial production of tetrapeptide-7. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. To this end, the present invention proposes an in-cell peptide and its application in the biological preparation of tetrapeptide-7. By fusing the in-cell peptide with tetrapeptide-7, high-purity tetrapeptide-7 can be produced efficiently and on a large scale, effectively avoiding the generation of byproducts and toxic and harmful substances.

[0006] In a first aspect, the present invention provides a Cth TerA inteptide variant, the amino acid sequence of which is shown in SEQ ID NO:3.

[0007] In this invention, the Cth TerA inteptide variant is obtained by targeted modification based on the original Cth TerA inteptide sequence.

[0008] In some embodiments of the present invention, the original sequence of the Cth TerA inteptide is a TerA gene fragment of Clostridium thermocellum ATCC27405.

[0009] In some embodiments of the present invention, the TerA gene fragment is selected from the fragment containing amino acid residues 68-400 in GenBank:WP_127837222.1.

[0010] A second aspect of the invention provides a nucleic acid molecule encoding a Cth TerA peptide variant described in the first aspect of the invention, the nucleotide sequence of which is shown in SEQ ID NO:4.

[0011] In some embodiments of the present invention, the nucleic acid molecule further includes a sequence having 85%, 90%, 95% or more identity with SEQ ID NO:4 and having the same self-cleavage activity.

[0012] In some embodiments of the present invention, the nucleic acid molecule further includes a sequence having 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4 and having the same self-cleavage activity.

[0013] A third aspect of the invention provides the application of the Cth TerA intapeptide variant described in the first aspect of the invention as a tag sequence in protein purification.

[0014] In this invention, the inventors discovered that the CthTerA inteptide variants after directionally modified exhibit stronger self-cleavage activity compared to the original CthTerA inteptide sequence, thereby achieving a higher self-cleavage effect.

[0015] A fourth aspect of the present invention provides a protein purification product, said protein purification product comprising at least one of the following (1) to (4):

[0016] (1) The Cth TerA peptide variant described in the first aspect of the present invention;

[0017] (2) The nucleic acid molecule described in the second aspect of the present invention;

[0018] (3) An expression cassette containing the nucleic acid molecules in (2);

[0019] Transformants containing nucleic acid molecules of (2) or expression cassettes of (3);

[0020] The transformants include viruses, bacteria, fungi, and cells.

[0021] In some embodiments of the present invention, the transformant is a bacterium.

[0022] In some embodiments of the present invention, the transformant is Escherichia coli.

[0023] A fifth aspect of the present invention provides a fusion protein comprising the Cth TerA intapeptide variant sequence and the tetrapeptide-7 sequence described in the preceding aspects.

[0024] In some embodiments of the present invention, the tetrapeptide-7 is GQPR.

[0025] In some embodiments of the present invention, the fusion protein further contains a modifying sequence.

[0026] In some embodiments of the present invention, the modified sequence includes, but is not limited to, at least one of the following: his tag sequence, GST tag sequence, Flag tag sequence, Halo tag sequence, HA tag sequence, Myc tag sequence, and Snap tag sequence.

[0027] In some embodiments of the present invention, the modification sequence is a his tag sequence.

[0028] In some embodiments of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO:5.

[0029] In some embodiments of the present invention, the nucleotide sequence of the fusion protein is shown in SEQ ID NO:6.

[0030] A sixth aspect of the present invention provides the use of the fusion protein described in the fifth aspect of the present invention in the preparation of tetrapeptide-7.

[0031] In this invention, the inventors constructed a tetrapeptide-7 expressing a CthTerA intapeptide variant tag based on the nucleotide sequence encoding the fusion protein, thereby achieving the technical effect of obtaining high-purity tetrapeptide-7 without using chemicals and enzymes.

[0032] A seventh aspect of the present invention provides a tetrapeptide-7 precursor substance, said tetrapeptide-7 precursor substance comprising at least one of the following (5) to (8):

[0033] (5) The fusion protein described in the fifth aspect of the present invention;

[0034] (6) The nucleic acid molecule encoding the fusion protein in (5);

[0035] (7) An expression cassette containing the nucleic acid molecules in (6);

[0036] (8) Transformants containing nucleic acid molecules from (6) or expression cassettes from (7);

[0037] The transformants include viruses, bacteria, fungi, and cells.

[0038] In some embodiments of the present invention, the transformant is a bacterium.

[0039] In some embodiments of the present invention, the transformant is Escherichia coli.

[0040] An eighth aspect of the present invention provides a method for preparing tetrapeptide-7 by biological means, comprising the following steps:

[0041] Connect the Cth TerA inner peptide variant sequence and the tetrapeptide-7 sequence described in the first aspect of the present invention, insert them into a plasmid vector, transfect them into transformants, induce protein expression with an inducer, collect the supernatant after cell lysis, perform autocleavage in a chromatography column, and collect the flow-through to obtain tetrapeptide-7.

[0042] In some embodiments of the present invention, the tetrapeptide-7 is GQPR.

[0043] In some embodiments of the present invention, the Cth TerA inteptide variant sequence is attached to the C-terminus of the tetrapeptide-7 sequence.

[0044] In some embodiments of the present invention, the Cth TerA intapeptide variant-tetrapeptide-7 fusion sequence fragment is inserted into a plasmid vector by a double enzyme digestion method using DNA ligase.

[0045] In some embodiments of the present invention, the DNA ligase is T4 DNA ligase.

[0046] Of course, those skilled in the art can also use other gene editing techniques to insert the Cth TerA intapeptide variant-tetrapeptide-7 fusion sequence into a plasmid vector to achieve the construction of a specific vector.

[0047] In some embodiments of the present invention, the nucleotide sequence of the constructed plasmid vector is shown in SEQ ID NO:7. In this invention, the inventors discovered that the inteptide Cth TerA-tetrapeptide-7 expression vector of the present invention has the effect of highly expressing the inteptide Cth TerA-tetrapeptide-7 fusion protein, and it can rapidly remove various tags based on the inteptide Cth TerA, thereby obtaining unmodified tetrapeptide-7 that can be directly used as a raw material.

[0048] In some embodiments of the present invention, positive clones are screened after transfection into transformants.

[0049] In some embodiments of the present invention, the screening is performed using kanamycin resistance plates.

[0050] In some embodiments of the present invention, the chromatography column is a Ni-NTA affinity chromatography column.

[0051] In some embodiments of the present invention, the specific operation of the self-cleavage is as follows: add buffer solution to the Ni-NTA affinity chromatography column, mix well, and incubate overnight at room temperature.

[0052] In some embodiments of the present invention, the buffer solution includes, but is not limited to, phosphate buffer.

[0053] In some embodiments of the present invention, the steps further include concentrating and drying the flowing liquid and the eluent.

[0054] In this invention, the inventors discovered that based on the above method, high-purity, unmodified tetrapeptide-7 can be obtained efficiently with a purity of over 85%.

[0055] Tetrapeptide-7 is an oligopeptide composed of four amino acids with the amino acid sequence GQPR. The inventors discovered that by utilizing the "central dogma" of mRNA translation into polypeptide chains in natural biological systems, amino acid addition can be achieved in an orderly manner according to gene coding information. This allows tetrapeptide-7 to be obtained by transferring genetically prepared material into a cellular system to obtain engineered organisms that stably express this genetic information product. Therefore, from a technical perspective, it has at least the following advantages compared to chemical synthesis: 1) Simple operation: once engineered bacteria expressing specific peptides are constructed, peptide expression can be continuously carried out; 2) Low cost: the substrates are all basic nutrients, requiring no expensive materials; 3) Fewer byproducts: easy purification, with virtually no synthetic byproducts, only metabolic products of the organism, which are easy to separate and purify; 4) Clean and environmentally friendly: almost pollution-free, meeting the demands of the "carbon neutrality" era; 5) High efficiency: highly efficient synthesis, allowing for large-scale production and acquisition. Therefore, it can be seen that the related applications in this invention have significant technical advantages compared to chemical synthesis. However, the problem with existing technologies is the lack of examples of tetrapeptide-7 biosynthesis. This is because the tetrapeptide-7 peptide chain is too short, making it difficult to detect and isolate in biological cultures. While some methods mention using oligopeptide fusion proteins containing protease cleavage sites to increase the length of the oligopeptide chain, making it easier to detect oligopeptide yield during expression and using affinity tags to reduce the difficulty of oligopeptide purification, this method requires the introduction of additional endonucleases after protein purification for oligopeptide release and separation. This increases the complexity of oligopeptide production and reduces oligopeptide yield. It also easily introduces additional amino acid residues, leading to changes in the performance or function of the oligopeptide. The integument in this invention effectively solves the above problems. Integuments are amino acid sequences capable of autocatalytic cleavage, allowing them to separate from the protein under specific conditions. The integument in this invention can achieve targeted cleavage of tetrapeptide-7, effectively removing various protein tags from specific proteins.

[0056] The beneficial effects of this invention are:

[0057] 1. This invention provides a novel Cth TerA inteptide variant sequence. By combining it with tetrapeptide-7 to form a fusion protein, a highly efficient and stable recombinant expression vector of tetrapeptide-7 is prepared after being introduced into a vector plasmid. After being transformed into bacteria to obtain engineered bacteria, high-purity unmodified tetrapeptide-7 can be obtained through induced expression.

[0058] 2. The tetrapeptide-7 in this invention is obtained based on the inteptide Cth TerA-tetrapeptide-7 recombinant expression vector. Its preparation method is simple, requiring only simple steps such as cell disruption and inteptide cleavage to efficiently obtain high-purity tetrapeptide-7. Moreover, the biosynthesis method can effectively reduce pollution and the generation of toxic and harmful byproducts, and the yield is large, making it suitable for large-scale industrial production of tetrapeptide-7, thereby obtaining high-quality tetrapeptide-7 with significant market value. Attached Figure Description

[0059] Figure 1 The results of activity tests on Cth TerA intima-peptides before and after directed evolution in this embodiment of the invention are shown.

[0060] Figure 2 This is an HPLC chromatogram of the production of tetrapeptide-7 from Cth TerA inteptide cleavage before and after directed evolution in an embodiment of the present invention. Detailed Implementation

[0061] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0062] Example 1: Construction of Cth TerA Intended Peptide Variants:

[0063] In this embodiment, the Cth TerA peptide is derived from the TerA gene of Clostridium thermocellum ATCC27405, and its original amino acid sequence is as follows:

[0064] QLALDTPIPTPDGWTTMGEIKAGDKVIDEKGRPCNVVAISEIDDTEQAYKINFRDGTSIVAGERHLWKVQVTNNGRREKLLTTGEMYQKQFKTKSKENRALFRIPIADAFILPENKLPIDPYLFGYWIGNGNAVKPEITVMRDDVDEVIKNIPYKLHNRYKQEGNSDILVYKELKSILVKNFREKRIPIEYLRASAQQRKRLLQGLIDSDGCVSTAKSQAIYVTILFELAKDVQDLLWSLGIKNTLKTAPSARYGIETGEICYLIKFTAFNDLEVSGLDRKLKRGRERNIKTRSHFHYIKSIEKTGKTKMRCIQVDSPSRLYLAGKSMIPTHN(SEQ ID NO:1).

[0065] Its corresponding nucleotide sequence is:

[0066] 5’-CAGCTGGCTCTGGATACTCCAATTCCAACTCCAGATGGTTGGACCACCATGGGCGAAATCAAAGCAGGTGATAAAGTGATCGACGAAAAAGGTCGTCCGTGCAACGTGGTGGCAATCTCTGAAATTGACGATACTGAACAGGCGTACAAAATTAACTTCCGTGATGGCACCTCCATCGTAGCGGGCGAACGTCATCTGTGGAAAGTGCAAGTTACCAATAACGGTCGTCGTGAAAAACTGCTGACCACCGGCGAAATGTATCAGAAACAGTTCAAAACCAAATCTAAAGAAAATCGTGCTCTGTTCCGTATCCCGATCGCCGATGCGTTCATCCTGCCAGAGAACAAACTGCCGATCGATCCGTACCTGTTTGGTTACTGGATCGGTAACGGTAACGCAGTTAAACCAGAAATCACTGTGATGCGTGACGACGTTGACGAAGTAATTAAAAACATTCCGTATAAACTGCACAACCGTTATAAACAGGAGGGTAACAGCGACATCCTGGTCTATAAAGAGCTGAAATCTATCCTGGTGAAAAACTTTCGTGAAAAGCGCATTCCGATCGAGTATCTGCGCGCTTCTGCTCAGCAGCGTAAACGTCTGCTGCAGGGTCTGATCGATTCCGATGGTTGTGTCTCTACTGCTAAATCTCAGGCAATCTATGTCACCATCCTGTTCGAGCTGGCTAAAGACGTACAGGACCTGCTGTGGTCCCTGGGTATTAAAAACACTCTGAAAACCGCGCCGAGCGCGCGTTATGGTATCGAGACCGGCGAAATCTGTTACCTGATCAAATTCACCGCATTCAACGATCTGGAGGTAAGCGGTCTGGATCGTAAACTGAAGCGTGGTCGTGAACGTAACATCAAAACTCGCAGCCACTTCCATTATATCAAATCTATTGAAAAAACCGGTAAAACCAAAATGCGTTGCATCCAGGTTGATTCTCCATCTCGTCTGTACCTGGCAGGTAAATCTATGATTCCTACCCACAAC-3’

[0067] (SEQ ID NO:2)。

[0068] The original sequence of the Cth TerA inte peptide was subjected to directed evolution using the phage-assisted continuous directed evolution system (PACE) (directed evolution operation referenced in Chinese Patent 202210830008.5), resulting in an evolved variant of the Cth TerA inte peptide with the following amino acid sequence:

[0069] QLSLDTPIPTPDGFTTMGEIKAGDKVIDEKGRPCNVVAISEIDDTEQAYKINFRDGTSIVAGERHLWQVQVTNNGRREKLLTTGEMYQKQFKTKSKENRALFRIPIADAFILPENKLPIDPYLFGYWIGNGNAVKPEITVMRDDVDEVIKNIPYKLHNRYKQEGNSDI LVYKELKSILVKNFREKRIPIEYLRASAQQRKRLLQGLIDSDGCVSTAKSQAIYVTILFELAKDVQDLLWSLGIKNTLKTAPSARYGIETGEICYLIKFTAFNDLEVSGLDRKLKRGRERNIKTRSHFHYIKSIEKTGKTKMRCIQVDSPSRLYLAGKSMIPTHN(SEQ ID NO:3).

[0070] The corresponding nucleotide sequence is:

[0071] 5’-CAGCTGTCTCTGGATACTCCAATTCCAACTCCAGATGGTTTCACCACCATGGGCGAAATCAAAGCAGGTGATAAAGTGATCGACGAAAAAGGTCGTCCGTGCAACGTGGTGGCAATCTCTGAAATTGACGATACTGAACAGGCGTACAAAATTAACTTCCGTGATGGCACCTCCATCGTAGCGGGCGAACGTCATCTGTGCAAAGTGCAAGTTACCAATAACGGTCGTCGTGAAAAACTGCTGACCACCGGCGAAATGTATCAGAAACAGTTCAAAACCAAATCTAAAGAAAATCGTGCTCTGTTCCGTATCCCGATCGCCGATGCGTTCATCCTGCCAGAGAACAAACTGCCGATCGATCCGTACCTGTTTGGTTACTGGATCGGTAACGGTAACGCAGTTAAACCAGAAATCACTGTGATGCGTGACGACGTTGACGAAGTAATTAAAAACATTCCGTATAAACTGCACAACCGTTATAAACAGGAGGGTAACAGCGACATCCTGGTCTATAAAGAGCTGAAATCTATCCTGGTGAAAAACTTTCGTGAAAAGCGCATTCCGATCGAGTATCTGCGCGCTTCTGCTCAGCAGCGTAAACGTCTGCTGCAGGGTCTGATCGATTCCGATGGTTGTGTCTCTACTGCTAAATCTCAGGCAATCTATGTCACCATCCTGTTCGAGCTGGCTAAAGACGTACAGGACCTGCTGTGGTCCCTGGGTATTAAAAACACTCTGAAAACCGCGCCGAGCGCGCGTTATGGTATCGAGACCGGCGAAATCTGTTACCTGATCAAATTCACCGCATTCAACGATCTGGAGGTAAGCGGTCTGGATCGTAAACTGAAGCGTGGTCGTGAACGTAACATCAAAACTCGCAGCCACTTCCATTATATCAAATCTATTGAAAAAACCGGTAAAACCAAAATGCGTTGCATCCAGGTTGATTCTCCATCTCGTCTGTACCTGGCAGGTAAATCTATGATTCCTACCCACAAC-3’(SEQ ID NO:4)。

[0072] The activity of the obtained CthTerA inte peptide variant was tested. The specific steps were as follows: the complete CthTerA inte peptide variant sequence was inserted into the GFP protein sequence. The presence of a complete GFP protein was observed to verify whether the CthTerA inte peptide variant possessed the self-cleavage activity expected of an inte peptide (GFP with an inserted foreign sequence is non-fluorescent, while when the foreign sequence has self-cleavage activity, it can achieve self-cleavage, excising itself from the GFP sequence, thus reverting to a complete GFP protein sequence and producing fluorescence). The original CthTerA inte peptide sequence was used as a control.

[0073] The results are as follows Figure 1 As shown.

[0074] It can be observed that the fluorescence intensity of the Cth TerA inte peptide evolutionary variant group is significantly stronger than that of the original Cth TerA inte peptide sequence group, indicating that the evolved Cth TerA inte peptide evolutionary variant has a higher self-cleavage efficiency compared to the original Cth TerA inte peptide sequence.

[0075] Example 2: Application of Cth TerA Integrity Evolutionary Variants in Tetrapeptide-7 Purification

[0076] To demonstrate the practical effectiveness of the Cth TerA intepitide variant in tetrapeptide-7 purification, the inventors conducted the following verification experiments.

[0077] A tetrapeptide-7 sequence was fused to the C-terminus of the Cth TerA in-peptide variant (SEQ ID NO:3), and the resulting amino acid sequence is as follows:

[0078] QLSLDTPIPTPDGFTTMGEIKAGDKVIDEKGRPCNVVAISEIDDTEQAYKINFRDGTSIVAGERHLWQVQVTNNGRREKLLTTGEMYQKQFKTKSKENRALFRIPIADAFILPENKLPIDPYLFGYWIGNGNAVKPEITVMRDDVDEVIKNIPYKLHNRYKQEGNSDILV YKELKSILVKNFREKRIPIEYLRASAQQRKRLLQGLIDSDGCVSTAKSQAIYVTILFELAKDVQDLLWSLGIKNTLKTAPSARYGIETGEICYLIKFTAFNDLEVSGLDRKLKRGRERNIKTRSHFHYIKSIEKTGKTKMRCIQVDSPSRLYLAGKSMIPTHNGQPR(SEQ ID NO:5).

[0079] The corresponding nucleotide sequence is:

[0080] 5’-CAGCTGTCTCTGGATACTCCAATTCCAACTCCAGATGGTTTCACCACCATGGGCGAAATCAAAGCAGGTGATAAAGTGATCGACGAAAAAGGTCGTCCGTGCAACGTGGTGGCAATCTCTGAAATTGACGATACTGAACAGGCGTACAAAATTAACTTCCGTGATGGCACCTCCATCGTAGCGGGCGAACGTCATCTGTGCAAAGTGCAAGTTACCAATAACGGTCGTCGTGAAAAACTGCTGACCACCGGCGAAATGTATCAGAAACAGTTCAAAACCAAATCTAAAGAAAATCGTGCTCTGTTCCGTATCCCGATCGCCGATGCGTTCATCCTGCCAGAGAACAAACTGCCGATCGATCCGTACCTGTTTGGTTACTGGATCGGTAACGGTAACGCAGTTAAACCAGAAATCACTGTGATGCGTGACGACGTTGACGAAGTAATTAAAAACATTCCGTATAAACTGCACAACCGTTATAAACAGGAGGGTAACAGCGACATCCTGGTCTATAAAGAGCTGAAATCTATCCTGGTGAAAAACTTTCGTGAAAAGCGCATTCCGATCGAGTATCTGCGCGCTTCTGCTCAGCAGCGTAAACGTCTGCTGCAGGGTCTGATCGATTCCGATGGTTGTGTCTCTACTGCTAAATCTCAGGCAATCTATGTCACCATCCTGTTCGAGCTGGCTAAAGACGTACAGGACCTGCTGTGGTCCCTGGGTATTAAAAACACTCTGAAAACCGCGCCGAGCGCGCGTTATGGTATCGAGACCGGCGAAATCTGTTACCTGATCAAATTCACCGCATTCAACGATCTGGAGGTAAGCGGTCTGGATCGTAAACTGAAGCGTGGTCGTGAACGTAACATCAAAACTCGCAGCCACTTCCATTATATCAAATCTATTGAAAAAACCGGTAAAACCAAAATGCGTTGCATCCAGGTTGATTCTCCATCTCGTCTGTACCTGGCAGGTAAATCTATGATTCCTACCCACAACGGTCAGCCGCGT-3’(SEQ ID NO:6)。

[0081] The sequence shown in SEQ ID NO:6 was biosynthesized by Guangzhou Aiji Biotechnology Co., Ltd. Of course, those skilled in the art can also choose other conventional methods in the art to synthesize the sequence according to the above composition, depending on the actual situation.

[0082] The 5' and 3' ends of the synthesized SEQ ID NO:6 were double-digested using BamHI and XhoI restriction enzymes, respectively, to obtain the digested fragment. Takara restriction endonucleases were used; please refer to the instruction manual for usage.

[0083] The blank pET28a plasmid was double-digested using the restriction enzyme sites BamHI and XhoI, and the corresponding fragments were recovered using 2% agarose gel to obtain the linearized pET28a plasmid vector.

[0084] The digested fragment and the linearized pET28a plasmid vector were ligated using T4 DNA ligase. The ligation method was performed in accordance with the pET28a plasmid instructions or conventional technical manuals in the field, resulting in a recombinant plasmid vector pET28a-included peptide variant-tetrapeptide-7 (nucleotide sequence as shown in SEQ ID NO:7) containing the integrapeptide variant-tetrapeptide-7 fusion sequence.

[0085] pET28a-in-peptide variant-tetrapeptide-7 was transformed into BL21(DE3) competent E. coli cells. The transformed E. coli were plated on solid LB agar plates containing kanamycin and incubated overnight at 37°C. Positive single clones were picked and cultured in LB liquid medium containing kanamycin with shaking, followed by sequencing to confirm successful transformation.

[0086] Positive clones were cultured in LB medium until the OD600 value reached 0.8. 1 / 20 volume of 1M Tris-HCl buffer (pH 8.5) and 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) were added to induce expression. The culture was continued at 37℃ and 200 rpm for 4-6 h. The cells were then collected by centrifugation at 4℃ and 10000 rpm for 20 min. The collected cells were washed twice with PBS.

[0087] The washed bacterial cells were resuspended in lysis buffer (composed of 20 mM Tris and 500 mM NaCl, pH 8.0) and the cells were lysed using pressure or sonication until no obvious cells were observed under microscopic staining. The cells were centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatant was collected and filtered through a 0.45 μm filter membrane to obtain the cell lysis filtrate.

[0088] After equilibrating the Ni-NTA affinity column with 20 column volumes of lysis buffer, add the filtered cell lysis buffer and transfer it into the Ni-NTA affinity column at a flow rate of 0.5 mL / min. Then wash thoroughly with 5 column volumes of lysis buffer containing 20 mM imidazole.

[0089] Add 50 mM phosphate buffer (pH 6.0) to the Ni-NTA affinity chromatography column, mix well, incubate at room temperature overnight, and collect the flow-through to obtain the purified tetrapeptide-7.

[0090] Use the original integrin sequence to replace the integrin variant sequence as a control.

[0091] The content and purity of the purified target protein (tetrapeptide-7) were determined using HPLC.

[0092] The results are as follows Figure 2 As shown.

[0093] It can be observed that after cleavage by the intapeptide variant, the content of tetrapeptide-7 in the flow solution can reach 0.85 mg / L of bacterial culture, with a purity of over 85%. This demonstrates that the pET28a-intapeptide variant-tetrapeptide-7 constructed in the embodiments of this invention can efficiently and rapidly obtain purified tetrapeptide-7.

[0094] 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 Cth TerA-containing peptide variant, characterized in that, The amino acid sequence of the Cth TerA-containing peptide variant is shown in SEQ ID NO:

3.

2. The application of the Cth TerA intapeptide variant of claim 1 as a tag sequence in protein purification.

3. A protein purification product, characterized in that, The protein purification product includes at least one of the following (1) to (4): (1) The Cth TerA-containing peptide variant of claim 1; (2) A nucleic acid molecule encoding the Cth TerA peptide variant of claim 1; (3) Expression cassettes containing nucleic acid molecules from (2); (4) Transformants containing nucleic acid molecules from (2) or expression cassettes from (3); The transformants include viruses, bacteria, fungi, and cells.

4. A fusion protein, characterized in that, The fusion protein contains the Cth TerA in-peptide variant sequence and the tetrapeptide-7 sequence as described in claim 1; wherein the tetrapeptide-7 is a GQPR oligopeptide.

5. The fusion protein according to claim 4, characterized in that, The fusion protein also contains a modification sequence; the modification sequence includes at least one of the following: his tag sequence, GST tag sequence, Flag tag sequence, Halo tag sequence, HA tag sequence, Myc tag sequence, and Snap tag sequence.

6. The use of the fusion protein according to any one of claims 4 to 5 in the preparation of tetrapeptide-7.

7. A tetrapeptide-7 precursor, characterized in that, The tetrapeptide-7 precursor includes at least one of the following (5) to (8): (5) The fusion protein according to any one of claims 4 to 5; (6) The nucleic acid molecule encoding the fusion protein in (5); (7) An expression cassette containing the nucleic acid molecules in (6); (8) Transformants containing nucleic acid molecules from (6) or expression cassettes from (7); The transformants include viruses, bacteria, fungi, and cells.

8. A method for preparing tetrapeptide-7 by biological means, comprising the following steps: A nucleic acid molecule encoding the Cth TerA containing a peptide variant and a tetrapeptide-7 sequence as described in claim 1 is inserted into a plasmid vector, transfected into transformants, and induced to express the protein. After cell lysis, the supernatant is collected, and the mixture undergoes autocleavage in a chromatography column. The collected flow-through yields tetrapeptide-7. The tetrapeptide-7 is a GQPR oligopeptide.

9. The method according to claim 8, characterized in that, The chromatography column includes a Ni-NTA affinity chromatography column.

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