Intrapeptide Ssp tau, its encoding gene and its application in the preparation of tetrapeptide-5

Abstract

This invention discloses an inteptide Ssp tau, characterized by its amino acid sequence as shown in SEQ ID No. 2. It also discloses its encoding gene and its application in the preparation of tetrapeptide-5. Furthermore, it discloses the Ssp tau-tetrapeptide-5 recombinant protein, the corresponding encoding gene, the expression vector, and the expression host bacteria. This invention discloses a novel inteptide Ssp tau and an inteptide Ssp tau-mediated tetrapeptide-5 expression and purification technique. Ssp tau and tetrapeptide-5 are combined to form a fusion protein, and the fusion protein gene sequence is constructed into PET-28a(+) to obtain a recombinant expression vector for tetrapeptide-5. Through simple steps such as induced expression, cell disruption, ultrafiltration, inteptide autocleavage, secondary ultrafiltration, and rotary lyophilization, high-purity tetrapeptide-5 can be obtained efficiently. This biosynthetic method is suitable for the industrial production of tetrapeptide-5 and has significant market value.

Description

Technical Field

[0001] This invention relates to an intima-peptide Ssp tau, its encoding gene, and its application in the preparation of tetrapeptide-5, belonging to the field of protein expression technology. Background Technology

[0002] Acetyl tetrapeptide-5, also known as eye peptide or eye serine peptide, is one of the earliest and most widely used skincare peptides. Acetyl tetrapeptide-5 is an inhibitor of angiotensin-converting enzyme (ACE), which inhibits the synthesis of angiotensin II, thereby relieving eye pressure and reducing capillary permeability, thus reducing the penetration of fluid from the blood into the periorbital tissues and effectively eliminating edema, eye bags, and dark circles. It has become a key ingredient in many skincare and cosmetic products. However, its enzymatic or chemical synthesis methods suffer from high costs, demanding reaction conditions, and numerous side reactions, making it unsuitable for large-scale industrial production. Furthermore, the small molecular weight of tetrapeptide-5 presents challenges in separation and purification, complex operations, and high costs, further hindering its large-scale application.

[0003] The development of molecular biology techniques has greatly promoted the expansion of the field of synthetic biology. Microbial synthesis technology, through the modification of natural microbial gene expression systems, can achieve efficient expression of exogenous peptides, and is expected to replace enzymatic catalysis and organic synthesis methods for preparing polypeptides. Peptide biosynthesis technology has many advantages: 1) No complex multi-step synthesis steps: After successfully constructing genetically engineered bacteria expressing the target protein through molecular biology techniques, peptide expression and production can be continuously carried out; 2) Low cost and mild operating conditions: the substrates involved in the synthesis are all basic nutrients, requiring no expensive materials or reaction equipment; 3) Few byproducts and easy purification: there are virtually no synthetic byproducts, no new synthetic intermediates are introduced, only products of biological metabolism, which are easy to separate and purify; 4) Environmentally friendly and sustainable development technology; 5) High synthesis efficiency and high product yield, enabling large-scale production of peptides.

[0004] However, there are currently no cases of producing tetrapeptide-5 using engineered microorganisms. Tetrapeptide-5 has a small molecular weight, making it difficult to isolate, purify, and detect from microbial fermentation products through direct expression. Fusion genes containing oligopeptides with self-cleavage sites can be constructed to extend the peptide chain length, making it easier to detect oligopeptide yield during expression, and affinity tags can be used to reduce the difficulty of oligopeptide purification. The discovery and modification of intepids can be effectively applied to the fusion expression and purification of short peptides. An intepid is a sequence present in a precursor protein. During the conversion of the precursor protein into a mature protein, the exopeptides at both ends of the intepid are linked by peptide bonds through self-splicing, while the intepid itself is released from the precursor protein. Intepids have wide applications in biotechnology, including protein linkage, protein cyclization, protein labeling, toxic protein expression, and the study of in vivo protein interactions. The use of intepids and their variants to mediate peptide chain purification and their application in large-scale peptide production is also attracting increasing attention. Therefore, there is a need in this field for a universal, easy-to-operate, and low-cost method for obtaining tetrapeptide-5. Summary of the Invention

[0005] This invention discloses an intimatinib peptide Ssp tau, characterized by its amino acid sequence as shown in SEQ ID No. 2.

[0006] The present invention also discloses the gene encoding the above-mentioned intipeptide Ssp tau.

[0007] Preferably, its nucleotide sequence is shown in SEQ ID No. 1.

[0008] The present invention also discloses a recombinant protein containing the peptide Ssp tau-tetrapeptide-5, characterized in that its amino acid sequence is shown in SEQ ID No. 4.

[0009] And the gene encoding the aforementioned inteptide Ssp tau-tetrapeptide-5 recombinant protein.

[0010] Preferably, its nucleotide sequence is shown in SEQ ID No. 3.

[0011] The present invention also discloses the expression vector of the above-mentioned in-containment peptide Ssp tau-tetrapeptide-5 recombinant protein.

[0012] Preferably, the carrier is a PET28a(+) plasmid.

[0013] The present invention also discloses the expression host bacteria of the above-mentioned in-container peptide Ssp tau-tetrapeptide-5 recombinant protein.

[0014] Preferably, the host bacterium is Escherichia coli.

[0015] The application of the aforementioned inteptide Ssp tau in the preparation of tetrapeptide-5.

[0016] This invention also discloses a method for expressing and purifying tetrapeptide-5 mediated by an internal peptide, characterized by the following steps:

[0017] (1) Construct an expression vector expressing the above-mentioned intimatinib Ssp tau-tetrapeptide-5, and transform the recombinant expression vector into the expression host bacteria to obtain recombinant engineered bacteria;

[0018] (2) Culture the recombinant engineered bacteria to induce the expression of exogenous recombinant proteins;

[0019] (3) Collect bacterial cells and disrupt them, centrifuge, collect the supernatant, dilute it and then ultrafilter it to collect the concentrate containing recombinant protein.

[0020] (4) Add self-cleavage reaction buffer to induce the N-terminus of the inteptide to cleave, and obtain a mixture of inteptide Ssp tau and tetrapeptide-5.

[0021] (5) Perform a second ultrafiltration, collect the filtrate, concentrate by rotary evaporation, and obtain purified tetrapeptide-5.

[0022] Preferably, the conditions for inducing expression in step (2) are as follows: the recombinant engineered bacteria are inoculated into LB medium and cultured until OD600 = 0.4-0.8, then IPTG is added to a final concentration of 0.1-5mM and the culture is continued to induce expression for 4-10 hours.

[0023] Preferably, in step (3), bacterial cells are collected by centrifugation.

[0024] Preferably, in step (3), cell disruption specifically refers to resuspending bacterial cells in lysis buffer, disrupting the cells using pressure or ultrasound, and then filtering the filtrate through a 0.45µm filter membrane.

[0025] Preferably, in step (3), a 10 kDa filter membrane is used for ultrafiltration; in step (5), a 3 kDa filter membrane is used for a second ultrafiltration (using a membrane pack or ultrafiltration tube) until the volume of the retained liquid is less than one-twentieth of the original volume. The filtrate passing through the filter membrane is collected, concentrated by rotary evaporation, and then freeze-dried to obtain tetrapeptide-5 powder.

[0026] This invention discloses a novel intron peptide Ssp tau and an Ssp tau-mediated tetrapeptide-5 expression and purification technique. A fusion protein is formed by combining Ssp tau and tetrapeptide-5, the amino acid sequence of which is shown in SEQ ID No. 4, and the DNA sequence in SEQ ID No. 3. The fusion protein gene sequence is constructed into PET-28a(+) to obtain a recombinant expression vector for tetrapeptide-5. Through simple steps including induction of expression, cell disruption, ultrafiltration, intron autocleavage, secondary ultrafiltration, and rotary lyophilization, high-purity tetrapeptide-5 can be obtained efficiently. This biosynthetic method is suitable for the industrial production of tetrapeptide-5 and has significant market value.

[0027] The beneficial effects of this invention are as follows: It discloses a novel integrin Ssp tau, which can be used to successfully express tetrapeptide-5, and experimental verification shows that the yield and stability can be guaranteed. The molecular weight of this integrin is approximately 15 kDa, while the molecular weight of tetrapeptide-5 is approximately 0.6 kDa. Due to the significant difference in molecular weight, they can be separated by ultrafiltration. This integrin-mediated peptide purification system purifies recombinant proteins by utilizing the self-cleavage function of the integrin, creating a method that eliminates the need for expensive resins and enzymes required for tag removal in traditional separation methods. This method is simple, rapid, efficient, and has low trial costs. Attached Figure Description

[0028] Figure 1 Schematic diagram of the construction of PET28a-Ssp tau-tetrapeptide-5 vector.

[0029] Figure 2 Schematic diagram of PET28a-Ssp tau-tetrapeptide-5 recombinant plasmid.

[0030] Figure 3 Agarose gel electrophoresis image of PCR-amplified Ssp tau-tetrapeptide-5 gene. Meaning of the bands in the image: M: marker, 1, 2: two replicates of the intapeptide Ssp tau-tetrapeptide-5.

[0031] Figure 4 E. coli expression of tetrapeptide-5 induces OD 600 Optimization, the meaning of the bands in the figure: 1: no induction, 2: induction at OD 0.4, 3: induction at OD 0.6, 4: induction at OD 0.8, 5: induction at OD 1.0.

[0032] Figure 5 Optimization of induction temperature for tetrapeptide-5 expression in Escherichia coli. The meanings of the bands in the figure are: 1: no induction, 2: induction at 25℃, 3: induction at 30℃, 4: induction at 37℃.

[0033] Figure 6The IPTG induction concentration for tetrapeptide-5 expression in Escherichia coli was optimized. The meanings of the bands in the figure are: 1: no induction, 2: 0.1 mM induction, 3: 0.5 mM induction, 4: 1 mM induction, 5: 2 mM induction, 6: 5 mM induction.

[0034] Figure 7 Optimization of induction time for tetrapeptide-5 expression in Escherichia coli. The meanings of the bands in the figure are: 1: no induction, 2: induction for 4 hours, 3: induction for 6 hours, 4: induction for 8 hours, and 5: induction for 10 hours.

[0035] Figure 8 Schematic diagram of HPLC detection of tetrapeptide-5. Detailed Implementation

[0036] The present invention will be further illustrated below through the construction and verification of a tetrapeptide-5 expression and isolation system. These specific embodiments should not be construed as limiting the scope of application of the present invention in any way. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0037] Example 1: Construction of the recombinant plasmid PET28a-Ssp tau-tetrapeptide-5

[0038] A fusion protein was constructed by combining Ssp tau and tetrapeptide-5. The amino acid sequence of Ssp tau is shown in SEQ ID No. 2, and its DNA sequence is shown in SEQ ID No. 1. The amino acid sequence of tetrapeptide-5 is shown in SEQ ID No. 5. Using plasmid PET28a and E. coli DH5α stored in our laboratory as templates, the gene encoding the intapeptide and the linearized PET28a(+) vector were cloned using the primers in Table 1. The tetrapeptide-5 gene fragment was synthesized, and the Ssp tau-tetrapeptide-5 gene fragment was amplified using Ssp tau and tetrapeptide-5 as templates. Amplification was performed using PrimerStar Max from TaKaRa, with PCR conditions of 94℃ for 10s, 55℃ for 10s, and 72℃ for 10s for 30 cycles. The plasmid backbone and the target gene fragment were ligated using a seamless cloning method. Using a seamless cloning kit, the PET28a(+) exoskeleton and the Ssp tau-tetrapeptide-5 gene fragment were mixed at a molar ratio of 1:3 and incubated in a 50℃ water bath for 20 min. The ligation product was transformed into *E. coli* DH5α competent cells, and the transformed cells were plated on agar plates containing 50 μg / mL kanamycin and cultured overnight. Single colonies that grew on the plates were picked and cultured overnight in 5 mL of LB medium containing 50 μg / mL kanamycin with shaking. Colony PCR was performed using universal primers for identification, and the plasmid was extracted and sequenced using universal primers. The sequencing results showed that the constructed PET28a-Ssp tau-tetrapeptide-5 sequence was correct. Figure 1 This is a schematic diagram of the plasmid construction process. The final constructed expression vector is shown below. Figure 2 As shown.

[0039] The primers used are shown in Table 1.

[0040] Table 1 Primer Table

[0041]

[0042] Example 2: Induced expression of fusion protein

[0043] The correctly sequenced recombinant vector was transformed into *E. coli* DH5α for plasmid amplification, and *E. coli* BL21(DE3) was used as the host bacterium for plasmid expression. The constructed PET28a-Ssp tau-tetrapeptide-5 recombinant plasmid is shown below. Figure 2 As shown, the Ssp tau-tetrapeptide-5 gene amplified by PCR was verified using agarose gel electrophoresis, and the results are as follows. Figure 3 As shown. After single colonies of *E. coli* BL21 were cultured overnight at 37°C, each colony was picked and incubated overnight at 37°C and 220 rpm in 5 ml of LB medium (containing KanR resistance). 5 ml of the bacterial culture was then incubated in 200 ml of TB medium at 37°C and 180 rpm until OD500 was reached. 600 After reaching a concentration of approximately 0.6-0.8, IPTG (final concentration 1 mM) was added, and the cells were cultured for another 24 hours. After 24 hours, the cells were collected by centrifugation at 3000 rpm, 4°C, for 15 minutes. The collected cells were washed with Tris buffer and then subjected to SDS-PAGE electrophoresis to detect protein expression levels. SDS-PAGE analysis: Coomassie staining SDS-PAGE analysis of lysate samples was performed in sample buffer (BioRadXT sample buffer + 4 mM TCEP). The lysate samples included induced and uninduced cells, as well as lysed and insoluble fractions of induced cells (the lysate samples were obtained by sonication and centrifugation in a buffer containing 25 mM sodium phosphate pH 5 buffer).

[0044] Example 3: Optimization of Induced Expression Conditions

[0045] (1) Confirmation of the timing of recombinant Escherichia coli induction of tetrapeptide-5 expression

[0046] After successful sequencing, PET28a-Ssp tau-tetrapeptide-5 transformants were selected and seeded into 5 mL of LB broth containing kanR resistance. 10 mL of LB broth was added at a 1% ratio. When the OD600 reached 0.4, 0.6, 0.8, and 1 respectively, IPTG was added to a final concentration of 1 mM. BL21 (empty vector) served as a control. All samples were incubated on a shaker at 37℃ and 220 rpm for 4 h. The precipitates were then centrifuged and SDS-PAGE was performed for verification. Figure 4 As shown.

[0047] (2) Optimization of induction temperature for tetrapeptide-5 expression induced by recombinant Escherichia coli

[0048] After confirming the sequencing results were correct, PET28a-Ssp tau-tetrapeptide-5 transformants were selected and seeded into 5 mL of LB liquid medium containing kanR resistance. 10 mL of LB liquid was added at a 1% ratio, and the optimal OD induction was performed according to the previous step. 600 Under the following conditions, IPTG was added to a final concentration of 1 mM, and induction was performed at 220 rpm for 4 h at 25℃, 30℃, and 37℃, respectively. The precipitates were then centrifuged and subjected to SDS-PAGE verification. Figure 5 As shown.

[0049] (3) Confirmation of the IPTG induction concentration for recombinant Escherichia coli tetrapeptide-5 expression

[0050] After confirming the sequencing results were correct, PET28a-Ssp tau-tetrapeptide-5 transformants were selected and seeded into 5 mL of LB liquid medium containing kanR resistance. 10 mL of LB liquid was added at a 1% ratio, and the optimal OD induction was performed according to the previous step. 600 The optimal induction temperature conditions were determined by adding IPTG to final concentrations of 0.1 mM, 0.5 mM, 1 mM, 2 mM, and 5 mM, respectively, and inducing at 220 rpm for 4 hours. The precipitates were then centrifuged and subjected to SDS-PAGE verification. Figure 6 As shown.

[0051] (4) Optimization of induction time for tetrapeptide-5 expression induced by recombinant Escherichia coli

[0052] After confirming sequencing accuracy, PET28a-Ssp tau-tetrapeptide-5 transformants were selected and seeded into 5 mL of LB broth containing kanR resistance. 10 mL of LB broth was added at a 1% ratio, and the optimal OD induction process was performed according to the previous steps. 600 The optimal induction temperature and optimal IPTG concentration were determined by induction at 220 rpm for 4 h, 6 h, 8 h, and 10 h, respectively. The precipitates were then centrifuged and subjected to SDS-PAGE verification. Figure 7 As shown.

[0053] Example 4: Tetrapeptide-5 was obtained by cleavage and ultrafiltration of the intima-containing peptide.

[0054] In this embodiment, we disclose the cleavage conditions of the intrinating peptide Ssp tau and the method for ultrafiltration purification to obtain tetrapeptide-5, the specific implementation of which is as follows:

[0055] The *E. coli* strain expressing the peptide-oligopeptide fusion protein was expanded to OD in LB medium. 600 Add 1 / 20 volume of 1M Tris-HCl buffer (pH 8.5) and IPTG to a final concentration of 0.5mM, and incubate at 37°C and 200 rpm for 4-6 hours. Centrifuge the culture at 10000 rpm at 4°C for 20 min to collect the bacterial cells. Wash the bacterial cells twice with PBS buffer. Resuspend the bacterial cells in lysis buffer (20mM Tris, 500mM NaCl, pH 8.0), and lyse the cells using a pressure lyser. Centrifuge at 12000 rpm at 4°C for 20 min, collect the supernatant, and filter through a 0.45µm filter membrane. The filtrate is the lysis buffer. Pass the lysis buffer through a 10kDa ultrafiltration tube until the cutoff volume is less than one-twentieth of the original volume. Repeat three times, collecting the concentrated solution containing the inteptide-tetrapeptide-5. Add one-third to one-tenth volume of cleaving buffer, mix well, and incubate overnight at 4°C. The concentrated solution was passed through a 3 kDa ultrafiltration tube until the cutoff volume was less than one-twentieth of the original volume. The filtrate passing through the membrane was collected. After rotary evaporation and concentration, the solution was freeze-dried to obtain tetrapeptide-5 powder. The powder was then reconstituted with water, and the content and purity of the reconstituted solution were determined by HPLC. The HPLC results are shown below. Figure 8 Ultrafiltration can be used to efficiently obtain high-purity tetrapeptide-5 with a purity of over 95%.

Claims

1. A peptide Ssp tau, characterized in that... Its amino acid sequence is shown in SEQ ID No.

2.

2. The gene encoding the intimatinib Ssp tau as described in claim 1.

3. The encoding gene according to claim 2, characterized in that... Its nucleotide sequence is shown in SEQ ID No.

1.

4. A recombinant protein containing the peptide Ssp tau-tetrapeptide-5, characterized in that... Its amino acid sequence is shown in SEQ ID No.

4.

5. The gene encoding the inteptide Ssp tau-tetrapeptide-5 recombinant protein as described in claim 4.

6. The encoding gene according to claim 5, characterized in that... Its nucleotide sequence is shown in SEQ ID No.

3.

7. An expression vector, characterized in that... The recombinant protein containing the peptide Ssp tau-tetrapeptide-5 as described in claim 4 is expressed.

8. The expression vector according to claim 7, characterized in that... The carrier is the PET28a(+) plasmid.

9. An expression host bacterium, characterized in that... The recombinant protein containing the peptide Ssp tau-tetrapeptide-5 as described in claim 4 is expressed.

10. The expression host bacterium according to claim 9, characterized in that... The host bacterium is Escherichia coli.

11. The use of the intimatinib Ssp tau according to claim 1 in the preparation of tetrapeptide-5.

12. A method for expressing and purifying tetrapeptide-5 mediated by an internal peptide, characterized in that... The steps include: (1) Construct an expression vector expressing the peptide Ssp tau-tetrapeptide-5 as described in claim 4, and transform the recombinant expression vector into the expression host bacteria to obtain recombinant engineered bacteria; (2) Cultivate the recombinant engineered bacteria to induce the expression of exogenous recombinant proteins; (3) Collect bacterial cells and disrupt them, centrifuge, collect the supernatant, dilute it and then ultrafilter it to collect the concentrated solution containing recombinant protein; (4) Add self-cleavage reaction buffer to induce the N-terminus of the inteptide to cleave, and obtain a mixture of inteptide Ssp tau and tetrapeptide-5. (5) Perform a second ultrafiltration, collect the filtrate, concentrate by rotary evaporation, and obtain purified tetrapeptide-5.

13. The method for expression and purification of tetrapeptide-5 mediated by an intrinating peptide according to claim 12, characterized in that... The conditions for inducing expression in step (2) are as follows: the recombinant engineered bacteria are inoculated into LB medium and cultured until OD500. 600 When the concentration is 0.4-0.8, add IPTG to a final concentration of 0.1-5 mM and continue inducing expression culture for 4-10 h.

14. The method for expression and purification of tetrapeptide-5 mediated by an intrinating peptide according to claim 12, characterized in that... In step (3), bacterial cells are collected by centrifugation.

15. The method for expression and purification of tetrapeptide-5 mediated by an intrinating peptide according to claim 12, characterized in that... In step (3), cell disruption specifically refers to resuspending bacterial cells in lysis buffer, disrupting the cells using pressure or ultrasound, and then filtering the filtrate through a 0.45 μm filter membrane.

16. The method for expression and purification of tetrapeptide-5 mediated by an intrinating peptide according to claim 12, characterized in that... In step (3), a 10 kDa filter membrane is used for ultrafiltration, and in step (5), a 3 kDa filter membrane is used for a second ultrafiltration.

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