A recombinant human plasminogen mutant and its preparation method and application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a recombinant human plasminogen mutant, its preparation method, and its application. A recombinant human plasminogen mutant, the amino acid sequence of which is shown in SEQ ID NO.3. The recombinant human plasminogen mutant of this invention, compared to wild-type mPLG, can be directly expressed solublely in the supernatant of *E. coli* fermentation broth. Soluble expression allows for direct chromatography of the fermentation broth to obtain the target protein, which has significant advantages over inclusion body renaturation treatment in terms of process simplification, activity loss, time cost, and production cost. Furthermore, the activity of the mutant protein was tested using the fibrin plate method and kit method. The results showed that the titer of the target protein was approximately 8 UI / mg, which is similar to the titer of mPLG obtained by renaturation before mutation and PLG obtained from human blood. In summary, compared to the wild type, the mutant strain exhibits significantly improved solubility while maintaining high protein activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a recombinant human plasminogen mutant, its preparation method, and its application. Background Technology

[0002] Plasminogen (PLG) is a functional protein found in human plasma, primarily synthesized by the liver, but expressed in all major organs. Its concentration in plasma is 0.06-0.25 g / L, making it a scarce plasma protein. PLG is an inactive zymogen that is converted into plasmin (PLM) by activators, thereby performing various physiological functions. As a broad-spectrum protease, PLG (PLM) participates in numerous physiological processes, including fibrin degradation, complement interaction, extracellular matrix degradation, cell migration, inflammation relief, collagen activation, and thrombolysis.

[0003] PLG is a single-chain serine protein composed of 791 amino-terminal sites, with a relative molecular weight of approximately 90 kDa. It includes seven domains: an N-terminal preactivation peptide domain (PAP), five highly repeating kringle domains (K1-K5), and a serine protease domain (SP). PLG is the most basic and core component of the fibrinolytic system, also known as the fibrinolytic system. This system refers to the process by which plasminogen activators act on the Arg560-Val561 peptide bond of PLG, converting PLG into PLM, which then degrades fibrinogen and other proteins. Commonly used activators include endogenous tissue plasminogen activator (tPA) and urokinase (uPA), as well as exogenous streptokinase (SK).

[0004] Thrombotic diseases are a major global health problem, and thrombolytic therapy is currently the main treatment strategy for thrombotic diseases. At present, the main thrombolytic drugs used clinically are plasminogen activators (PLGs), including t-PA, uPA, and SK. These drugs convert plasminogen activator (PLG) into plasminogen activator (PLM) to exert their effects. Although they show good thrombolytic efficacy, they still have problems such as bleeding risk and low efficiency in dissolving old thrombi. PLG, as an inactive proenzyme form, is not only easily distributed in the body but also has a low bleeding risk and participates widely in various physiological activities. Furthermore, PLG can be used to treat plasminogen deficiency, woody conjunctivitis, and other diseases. Therefore, PLG has broad pharmaceutical prospects and research value.

[0005] However, current research and applications of PLG mainly rely on human plasma extraction, which is strictly limited by plasma resources. Recombinant protein expression offers the possibility of large-scale production, and it also exhibits unique advantages in sequence design, functional optimization, and targeted modification. In recent years, much work has focused on PLG recombinant expression, with multiple attempts made in Escherichia coli, Pichia pastoris, and mammalian cells. Escherichia coli, as the most economical and efficient expression system, has become a research focus. However, unfortunately, both full-length PLG and truncated mutants such as mPLG and μPLG containing only the core functional domain are expressed in inclusion bodies in E. coli, requiring renaturation treatment to obtain the target protein. This not only complicates the production process, but the inefficient and time-consuming renaturation process may also lead to decreased protein activity and yield, while increasing production costs. These problems severely limit the research and development of PLG in the E. coli system, further restricting its further development in biomedical research and clinical applications. Summary of the Invention

[0006] To address the technical problem that existing PLG is expressed in E. coli as inclusion bodies, requiring renaturation treatment to obtain the target protein, which not only complicates the production process, but also leads to low efficiency and long renaturation process, resulting in decreased protein activity and yield, but also increases production costs, this invention proposes a recombinant human plasminogen mutant, its preparation method, and its application.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] Firstly, a recombinant human plasminogen mutant, the amino acid sequence of which is shown in SEQ ID NO.3.

[0009] Specifically, amino acids 544-791 were truncated from the wild-type PLG sequence, which is 791 amino acids in length. The sequence is shown in SEQ ID NO.2, which is the truncated version of PLG, mPLG. Site-directed amino acid mutations were performed on the mPLG sequence to obtain the recombinant human plasminogen mutant. The amino acid mutation sites include H569N, V577I, P609S, R637K, K645A, V657I, F681Y, and Q721K.

[0010] Secondly, a nucleotide encoding the above-mentioned recombinant human plasminogen mutant, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0011] Thirdly, a method for preparing a recombinant human plasminogen mutant includes the following steps: synthesizing a DNA sequence with an amino acid sequence as shown in SEQ ID NO.3 using gene synthesis methods, optimizing the codons using Escherichia coli as the host, cloning it into a vector, and constructing a mutant strain (mPLG-8P); transforming it into competent cells, inducing and expressing the mutant strain, harvesting the fermentation broth, and obtaining the target recombinant human plasminogen mutant through affinity chromatography.

[0012] Furthermore, the vector is a pET sequence vector. Even further, the pET sequence vector is selected from the pET30a vector.

[0013] The mutant strain exhibits significantly improved solubility in Escherichia coli, allowing the target protein to be purified directly from the fermentation broth supernatant.

[0014] Fourthly, the application of the recombinant human plasminogen mutant mentioned in the first aspect above in the preparation of drugs for the prevention or treatment of thrombosis.

[0015] Fifthly, the application of the recombinant human plasminogen mutant mentioned in the first aspect above in the preparation of drugs for the prevention or treatment of plasminogen deficiency.

[0016] The sixth aspect concerns the application of the recombinant human plasminogen mutant mentioned in the first aspect in the preparation of drugs for the prevention or treatment of woody conjunctivitis.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The recombinant human plasminogen mutant mPLG-8P provided in this application introduces amino acid mutations into the truncated amino acid sequence of the mutant mPLG. The mutation sites include H569N, V577I, P609S, R637K, K645A, V657I, F681Y, and Q721K. Expression analysis shows that this recombinant human plasminogen mutant mPLG-8P, compared to wild-type mPLG, can be directly expressed solublely in the supernatant of E. coli fermentation broth. Soluble expression allows for direct chromatography of the fermentation broth to obtain the target protein. Compared to inclusion body renaturation treatment (where mPLG before mutation is expressed as inclusion bodies and requires renaturation to obtain the target protein), this method offers significant advantages in terms of process simplification, activity loss, time cost, and production cost. Meanwhile, the activity of the mutant protein was tested using the fibrin plate method and a kit method. The results showed that the titer of the target protein was approximately 8 UI / mg, which was similar to that of mPLG obtained by renaturation before mutation and PLG obtained from human blood. Overall, compared with the wild type, the mutant strain showed significantly improved solubility while maintaining high protein activity. Attached Figure Description

[0019] To more clearly illustrate the solutions in this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 Analysis and comparison of the amino acid sequences of human plasminogen with those of plasminogen from multiple other species;

[0021] Figure 2 A schematic diagram of the sequence modification of the human plasminogen mutant mPLG-8P;

[0022] Figure 3 SDS-PAGE analysis of mPLG-8P fermentation supernatant;

[0023] Figure 4 SDS-PAGE analysis of mPLG-8P chromatography experiment;

[0024] Figure 5 To determine the protein activity of mPLG-8P using the fibrin plate method. Detailed Implementation

[0025] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0026] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0027] Example 1: Construction of plasminogen mutant strain

[0028] The amino acid sequences of human plasminogen (PLG) were searched in the NCBI online database. A total of nine PLG samples from different sources were selected: human (>AAA60113.1), rhesus macaque (NP_001036540.1), longhorn beetle (>XP_066228882.1), greater silver-striped bat (>XP_066104612.1), ditch rat (>CAB46014.1), house mouse (AAA50168.1), Nicobar pigeon (>XP_065489086.1), painted turtle (XP_008162213.3), and rainbow trout (>NP_001117863.1). These PLG samples were analyzed and aligned using the online amino acid sequence analysis website CLUSTALW (https: / / www.genome.jp / tools-bin / clustalw) (the sequence alignment results only extracted the latter half of the core catalytic region). The results are as follows: Figure 1 As shown. Sequence alignment revealed that human PLG differs from PLG from other sources in the conservation of some amino acid residues. A total of 8 amino acid sites were selected, namely H569, V577, P609, R637, K645, V657, F681, and Q721.

[0029] This embodiment uses the full-length PLG as a base, extracting its core functional domains to construct the mutant strain mPLG; then, eight amino acid mutations are introduced into mPLG, including H569N, V577I, P609S, R637K, K645A, V657I, F681Y, and Q721K, to construct the mutant strain mPLG-8P, whose amino acid sequence is shown in SEQ ID NO.3. A schematic diagram of the mutant strain construction is shown below. Figure 2 As shown.

[0030] The amino acid sequence of the full-length PLG is shown in SEQ ID NO.1, as follows:

[0031] MEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN。

[0032] The amino acid sequence of the mutant mPLG is shown in SEQ ID NO.2 as follows:

[0033] MSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACL PSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN.

[0034] The amino acid sequence of the mutant strain mPLG-8P is shown in SEQ ID NO.3, as follows:

[0035] MSFDCGKPQVEPKKCPGRVVGGCVANPHSWPWQISLRTRFGMHFCGGTLISPEWVLTAAHCLEKSSRPSSYKVILGAHQEVNLEPHVQEIEVSKLFLEPTRADIALLKLSSPAVIITDKVIPACL PSPNYVVADRTECYITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVKSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN.

[0036] Example 2: Expression and identification of recombinant plasminogen mutant

[0037] The DNA sequence of mPLG-8P was synthesized using gene synthesis methods, and codon optimization was performed using *E. coli* as the host. It was then cloned into the pET30a vector to construct the recombinant plasmid mPLG-8P-pET30a. The recombinant plasmid was transformed into BL21(DE3) competent cells, and positive single clones were picked and inoculated into 10 mL of LB medium (containing kanamycin at a final concentration of 50 μg / mL) and cultured overnight at 37°C. The cells were then transferred to fresh LB medium at a 1% inoculation rate and cultured until the OD value (absorbance) reached 0.6-0.8. IPTG (isopropyl-β-D-thiogalactopyranoside) was added at a final concentration of 1 mM, and expression was induced at 23°C for 12-15 h. The cells were collected by centrifugation at 6000 rpm for 10 min for further processing or temporarily stored at -20°C. The bacterial cells were disrupted and centrifuged (12000 rpm, 2 min). Equal volumes of the supernatant from mPLG and mPLG-8P were directly analyzed by SDS-PAGE (sodium dodecyl sulfonate-polyacrylamide gel electrophoresis). Results are as follows: Figure 3 As shown, mPLG was almost entirely in the precipitate, while most of the mPLG-8P protein was in the supernatant, with only a small portion in the precipitate. Therefore, the solubility of mPLG-8P expressed in E. coli was significantly improved after mutation at eight amino acid sites.

[0038] In this embodiment, the nucleotide sequence encoding the above-mentioned recombinant human plasminogen mutant mPLG-8P is shown in SEQ ID NO.4, and is as follows:

[0039] Example 3: Purification of recombinant plasminogen mutant

[0040] The bacterial culture from Example 2, after centrifugation, was resuspended in PBS, homogenized using a high-pressure homogenizer, and the supernatant after high-speed centrifugation and filtration was subjected to affinity chromatography using Ni-NTA packing material with polyacrylate microspheres as the matrix. The loading buffer contained a gradient of 5-10 mM imidazole and 50-500 mM imidazole to elute the target protein mPLG-8P. The content and purity of mPLG protein in each peak were detected by SDS-PAGE gel electrophoresis. The collected target protein eluent was dialyzed through a 10 kDa ultrafiltration concentrator to replace the buffer and concentrate the target protein mPLG-8P. The purity of the purified recombinant human plasminogen mutant mPLG-8P was detected by SDS-PAGE. The SDS-PAGE gel electrophoresis results are shown below. Figure 4 As shown, the target protein was eluted at concentrations of 50, 75, 100, 150, and 300 mM imidazole. The molecular weight was around 30 kDa. Based on the band, the purity was estimated to be over 90%, and there were no other impurities, indicating that the target protein had very high purity.

[0041] Example 4: Detection of mPLG-8P activity and function using the fibrin plate method

[0042] Plasminogen is an inactive zymogen form that requires an activator to become active. In this example, urokinase was used as the activator to perform a fibrin plate test to determine fibrinolytic activity.

[0043] Preparation of fibrin plates: Add 0.75% w / v agarose (0.15g) and 150mM NaCl (0.1755g) to an Erlenmeyer flask. Measure 20mL of Tris-HCl buffer (20mM Tris, pH 8.0) and mix thoroughly. Seal the Erlenmeyer flask with breathable sealing film and heat in a microwave oven. When the solution becomes clear, remove it and cool until it is no longer hot to the touch. Add 100μL of 100U / mL thrombin solution and 1mL of 10mg / mL fibrinogen solution to the flask. Shake the Erlenmeyer flask to mix thoroughly, avoiding the formation of air bubbles. Then pour the mixture into a Petri dish and let it stand at room temperature for about 20 minutes to solidify and form fibrin plates. In this step, pay close attention to the temperature of the mixed solution before adding the thrombin and fibrinogen solutions. If the temperature is too high, white flocculent matter will form; if the temperature is too low, premature solidification will occur.

[0044] Oxford cups were placed on the prepared fibrin plates. 40 μL of PBS buffer containing 10 μL of 50 IU / mL urokinase, 20 μg of blood-derived PLG, mPLG (expressed in *E. coli*), mPLG-8P, and mPLG (expressed in *Pichia pastoris*) were added to each well. The plates were incubated overnight at 37°C. The size of the degradation zones of each sample was compared to determine its fibrinolytic activity. The results are shown below. Figure 5As shown, sample 1 was PLG (human blood-derived), sample 2 was mPLG (expressed in *E. coli*), sample 3 was mPLG-8P, sample 4 was PBS, and sample 5 was mPLG (expressed in *Pichia pastoris*). The results showed that the degradation zone produced by the recombinant mutant strain mPLG-8P expressed in *E. coli* was smaller than that produced by the blood-derived PLG. Therefore, it was determined that the fibrinolytic activity of the mutant strain mPLG-8P was lower than that of the blood-derived PLG after urokinase activation.

[0045] Furthermore, the potency of human PLG and mPLG-8P was tested. The results showed that the potency of human PLG was approximately 10 UI / mg, while that of mPLG-8P (expressed in E. coli) was approximately 9 UI / mg and 8 UI / mg, with mPLG-8P having a slightly lower potency than human PLG. However, the solubility of the mutant strain mPLG-8P was significantly improved, solving the problem of insolubility of recombinant PLG expressed in E. coli, thus demonstrating great significance and potential.

[0046] Obviously, the embodiments described above are only some embodiments of this application, not all embodiments. The accompanying drawings show preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms; rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this application's specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this application.

Claims

1. A recombinant human plasminogen mutant, characterized in that, The nucleotide sequence is shown as SEQ ID NO.

4.

2. The recombinant human plasminogen mutant of claim 1, wherein, The molecular weight of the recombinant human plasminogen mutant is 30KDa.

3. A nucleotide encoding the recombinant human plasminogen mutant of claim 1, wherein, The nucleotide sequence is shown as SEQ ID NO.

4.

4. A method for producing a recombinant human plasminogen mutant, characterized by, It comprises the following steps: The DNA sequence with the nucleotide sequence shown as SEQ ID NO. 4 is synthesized by using the method of gene synthesis, and is codon-optimized with E. coli as the host, cloned into a vector, and constructed into a mutant strain; the mutant strain is transformed into a competent cell, induced and expressed, the fermentation broth is harvested, and the target recombinant human plasminogen mutant is obtained through affinity chromatography.

5. The method for preparing the recombinant human plasminogen mutant according to claim 4, characterized in that, The vector is a pET sequence vector.

6. The method of producing a recombinant human plasminogen mutant according to claim 5, wherein, The pET sequence vector is selected from a pET30a vector.

7. The use of the recombinant human plasminogen mutant of claim 1 in the preparation of a medicament for treating plasminogen deficiency.

8. The use of the recombinant human plasminogen mutant of claim 1 in the preparation of a medicament for treating xerophthalmia.

Images