A modified fusion protein and its application

Abstract

This application relates to the fields of genetic engineering and enzyme engineering technology, specifically to a modified fusion protein and its application. The modified fusion protein is obtained by linking maltose-binding protein and heparinase I with linker peptides of different lengths, which improves the thermal stability, activity, and catalytic specificity constant of the maltose-binding protein fusion with heparinase I, and can be used to produce low molecular weight heparin, greatly increasing its industrial application value.

Description

Technical Field

[0001] This application relates to the fields of genetic engineering and enzyme engineering technology, specifically to a modified fusion protein and its applications. Background Technology

[0002] Heparinase I (HepI, EC 4.2.2.7) is an important polysaccharide lyase that catalyzes the cleavage of heparin. One of the most important uses of heparinase I is the preparation of low molecular weight heparin, an important anticoagulant drug. In addition, heparinase I has broad scientific research and clinical application value, including elucidating the fine structure and biological function of heparin polysaccharide molecules, quality control of heparin and low molecular weight heparin, and as a component of in vitro coagulation assay kits. Given the important scientific research and clinical application value of heparinase I, researchers have long studied its recombinant expression. Although heterologous soluble expression of heparinase I has been achieved and commercial products exist, large-scale industrial application has not yet been realized, with the key bottleneck being its poor thermostability. Currently, the fusion enzyme of maltose-binding protein (MBP) linked by a natural linker peptide to heparinase I has achieved highly efficient intracellular soluble expression in *E. coli*, but its half-life at 30°C is only 10 min (Kuang Y, Xing XH, Chen Y, Ye F, Chen Y, Yan Y, et al. Production of heparin oligosaccharides by fusion protein of MBP–heparinase I and the enzyme thermostability. *J Mol Catal B Enzym.* 2006; 43:90–95.). To improve the thermostability of heparinase I, Chen et al. increased the half-life of MBP-HepI at 30°C by 35% through site-directed mutagenesis and by adding Ca during enzyme production. 2+This further improved the stability of the fusion enzyme (Chen S, Huang Z, Wu J, Chen Y, Ye F, Zhang C, et al. Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I inrecombinant Escherichia coli. Appl Microbiol Biotechnol. 2013; 97:2907–16.). However, their method of directly modifying the heparinase I site in the fusion enzyme had limited effect on improving its thermostability, and there is an urgent need to develop other technologies to enhance the thermostability of fused heparinases.

[0003] Numerous studies have shown that linker peptides can influence the expression and catalytic properties of fusion enzymes. Lu et al. introduced different linker peptides into the fusion enzymes of β-glucanase (Glu) and xylanase (Xyl). They found that when (GGGGS)2 was used as the linker peptide, the catalytic efficiency of Glu and Xyl in the fusion enzyme was increased by 326% and 43%, respectively, compared to the parent protein; while when (EAAAK)3 was used as the linker peptide, the catalytic efficiency was increased by 262% and 31%, respectively, and the thermal stability of Glu was improved compared to before fusion (Lu P, Feng MG. Bifunctional enhancement of abeta-glucanasexylanase fusion enzyme by optimization of peptide linkers. Appl Microbiol Biotechnol. 2008; 79:579–87.). Patent CN 105753945 B describes an improvement in the half-life of MBP-fused heparinase I from 10 min to approximately 35 min by replacing the native linker peptides encoded on the commercially available pMAL-c2X plasmid with (GGGGS)5 and (EAAAK)5. These examples demonstrate that suitable linker peptides can enhance the target properties of fusion enzymes. Summary of the Invention

[0004] This application provides a linker peptide. The modified fusion protein formed by linking MBP and heparinase I with this linker peptide can significantly improve thermal stability, activity, and catalytic specificity constant. The specific scheme is as follows:

[0005] 1. A modified fusion protein, wherein the amino acid sequence is selected from one of the following:

[0006] SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

[0007] 2. A nucleic acid molecule encoding a fusion protein as described in item 1.

[0008] 3. A vector comprising the nucleic acid molecule as described in item 2.

[0009] 4. A genetically engineered bacterium comprising the vector described in item 3.

[0010] 5. The application of the fusion protein described in item 1 in the production of low molecular weight heparin.

[0011] 6. A method for producing low molecular weight heparin, comprising using a fusion protein as described in item 1, a nucleic acid molecule as described in item 2, a vector as described in item 3, or a genetically engineered bacterium as described in item 4.

[0012] Beneficial effects:

[0013] In this application, a modified fusion protein is obtained by linking maltose-binding protein and heparinase I with linker peptides of different lengths. This improves the thermal stability, activity, and catalytic specificity constant of the maltose-binding protein-heparinase I fusion. The maltose-binding protein-heparinase I fusion, achieving the desired improved properties, can be used to produce low molecular weight heparin, significantly increasing its industrial application value. Simultaneously, it can promote the upgrading of the heparin drug industry chain, contribute to the development of the biopharmaceutical industry, and provide insights for the application development of other industrial enzyme systems. Attached Figure Description

[0014] Figure 1-1 The image shows an SDS-PAGE analysis of the MBP fusion heparinase I expressed by Escherichia coli TB1 with linker peptides F1, F2, and F3 in Example 1 of this application. In this image, band 1 is the crude enzyme solution of the fusion protein MBP-F1-HepI, band 2 is the purified MBP-F1-HepI, band 3 is the crude enzyme solution of the fusion protein MBP-F2-HepI, band 4 is the purified MBP-F2-HepI, band 5 is the crude enzyme solution of the fusion protein MBP-F3-HepI, band 6 is the purified MBP-F3-HepI, band M is the protein molecular weight standard, and the arrows indicate the location of the target fusion protein band.

[0015] Figure 1-2The image shows an SDS-PAGE analysis of the MBP fusion heparinase I expressed by Escherichia coli TB1 with linker peptides F4, F5, and F6 in Example 1 of this application. In the image, band 7 is the crude enzyme solution of the fusion protein MBP-F4-HepI, band 8 is the purified MBP-F4-HepI, band 9 is the crude enzyme solution of the fusion protein MBP-F5-HepI, band 10 is the purified MBP-F5-HepI, band 11 is the crude enzyme solution of the fusion protein MBP-F6-HepI, band 12 is the purified MBP-F6-HepI, band M is the protein molecular weight standard, and the arrows indicate the location of the target fusion protein band.

[0016] Figure 2 The specific activity of the modified fusion proteins with different linker peptides in Example 2 of this application is shown;

[0017] Figure 3-1 The diagram shows the time curves of the residual activity of the modified fusion proteins with different linking peptides in Example 3 of this application at 30°C.

[0018] Figure 3-2 The diagram shows the time curves of the residual activity of the modified fusion proteins with different linking peptides in Example 3 of this application at 35°C. Detailed Implementation

[0019] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0020] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0021] In this application, a linker peptide refers to an amino acid sequence used to link two or more target proteins or protein domains, typically ranging from 3 to 50 amino acids in length. Linker peptides can be used to link two or more proteins, peptides, antibodies, etc., in different systems, and those skilled in the art can select an appropriate linker peptide based on the properties of the proteins, peptides, or antibodies to be linked.

[0022] In this application, the amino acid sequence of the linker peptide F1 is: GGGGS (SEQ ID NO:1).

[0023] The amino acid sequence of the linker peptide F2 is: (GGGGS)2 (SEQ ID NO:2).

[0024] The amino acid sequence of the linker peptide F3 is: (GGGGS)3 (SEQ ID NO:3).

[0025] The amino acid sequence of the linker peptide F4 is: (GGGGS)4 (SEQ ID NO:4).

[0026] The amino acid sequence of the linker peptide F6 is: (GGGGS)6 (SEQ ID NO:5).

[0027] There are no particular restrictions on the gene encoding the linker peptide, as long as the corresponding linker peptide sequence can be expressed through translation. For example, when the linker peptide is F1, its nucleotide sequence can be as shown in SEQ ID NO:6, and its nucleotide sequence is as follows:

[0028] GGTGGTGGCGGCAGC (SEQ ID NO:6).

[0029] When the linker peptide is F2, its nucleotide sequence can be as shown in SEQ ID NO:7, and its nucleotide sequence is as follows:

[0030] GGTGGTGGCGGCAGCGGTGGCGGCGGTAGC (SEQ ID NO: 7).

[0031] When the linker peptide is F3, its nucleotide sequence can be as shown in SEQ ID NO:8, and its nucleotide sequence is as follows:

[0032] GGTGGTGGCGGCAGCGGTGGCGGCGGTAGCGGCGGTGGTGGAT CC (SEQ ID NO: 8).

[0033] When the linker peptide is F4, its nucleotide sequence can be as shown in SEQ ID NO:9, and its nucleotide sequence is as follows:

[0034] GGTGGTGGCGGCAGCGGTGGCGGCGGTAGCGGCGGTGGTGGATC CGGTGGCGGTGGTTCT(SEQ IDNO:9)

[0035] When the linker peptide is F6, its nucleotide sequence can be as shown in SEQ ID NO:10, and its nucleotide sequence is as follows:

[0036] GGTGGTGGCGGCAGCGGTGGCGGCGGTAGCGGCGGTGGTGGAT CCGGTGGCGGTGGTTCTGGTGGTGGTGGCAGCGGTGGTGGCGGCAG C (SEQ ID NO: 10).

[0037] This application provides a fusion protein selected from one of the following:

[0038] MBP-F1-HepI, MBP-F2-HepI, MBP-F3-HepI, MBP-F4-HepI, MBP-F6-HepI.

[0039] In this application, when the fusion protein is MBP-F1-HepI, it has the amino acid sequence shown in SEQ ID NO:11.

[0040] SEQ ID NO:11:

[0041] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGGGGSQQKKSGNIPYRVNVQADSAKQSEIIDNKWVAVGINKPYALQYDDKLRFNGKPSYRFELKAEDNSLEGYAAGETKGRIELSYSYATTNDFKKFPPSVYQNAQKLKTVYHYGKGICEQGSSRSYTFSVYIPSSFPDNATTIFAQWHGAPSRTLVATPEGEIKTLSIEEFLALYDRMIFKKNIAHDKVEKKDKDGKITYVAGKPNGWKVEQGGYPPLAFGFSKGYFYIKANSDRQWLTDKADRNNANPENSEVMKPYSSEYKTSTIAYKMPFAQFPKDCWITFDVAIDWTKYGKEANTILKPGKLDVMMTYTKNKKPQKAHIVNQQEILIGRNDDDGYYFKFGIYRVGNSTVPVTYNLSGYSETAR

[0042] In the present application, when the fusion protein is MBP-F2-HepI, it has the amino acid sequence shown in SEQ ID NO: 12.

[0043] SEQ ID NO: 12:

[0044] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGGGGSGGGGSQQKKSGNIPYRVNVQADSAKQSEIIDNKWVAVGINKPYALQYDDKLRFNGKPSYRFELKAEDNSLEGYAAGETKGRIELSYSYATTNDFKKFPPSVYQNAQKLKTVYHYGKGICEQGSSRSYTFSVYIPSSFPDNATTIFAQWHGAPSRTLVATPEGEIKTLSIEEFLALYDRMIFKKNIAHDKVEKKDKDGKITYVAGKPNGWKVEQGGYPPLAFGFSKGYFYIKANSDRQWLTDKADRNNANPENSEVMKPYSSEYKTSTIAYKMPFAQFPKDCWITFDVAIDWTKYGKEANTILKPGKLDVMMTYTKNKKPQKAHIVNQQEILIGRNDDDGYYFKFGIYRVGNSTVPVTYNLSGYSETAR

[0045] In the present application, when the fusion protein is MBP-F3-HepI, it has the amino acid sequence shown in SEQ ID NO: 13.

[0046] SEQ ID NO: 13:

[0047] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGGGGSGGGGSGGGGSQQKKSGNIPYRVNVQADSAKQSEIIDNKWVAVGINKPYALQYDDKLRFNGKPSYRFELKAEDNSLEGYAAGETKGRIELSYSYATTNDFKKFPPSVYQNAQKLKTVYHYGKGICEQGSSRSYTFSVYIPSSFPDNATTIFAQWHGAPSRTLVATPEGEIKTLSIEEFLALYDRMIFKKNIAHDKVEKKDKDGKITYVAGKPNGWKVEQGGYPPLAFGFSKGYFYIKANSDRQWLTDKADRNNANPENSEVMKPYSSEYKTSTIAYKMPFAQFPKDCWITFDVAIDWTKYGKEANTILKPGKLDVMMTYTKNKKPQKAHIVNQQEILIGRNDDDGYYFKFGIYRVGNSTVPVTYNLSGYSETAR

[0048] In the present application, when the fusion protein is MBP-F4-HepI, it has the amino acid sequence shown in SEQ ID NO:14.

[0049] SEQ ID NO:14:

[0050] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGGGGSGGGGSGGGGSGGGGSQQKKSGNIPYRVNVQADSAKQSEIIDNKWVAVGINKPYALQYDDKLRFNGKPSYRFELKAEDNSLEGYAAGETKGRIELSYSYATTNDFKKFPPSVYQNAQKLKTVYHYGKGICEQGSSRSYTFSVYIPSSFPDNATTIFAQWHGAPSRTLVATPEGEIKTLSIEEFLALYDRMIFKKNIAHDKVEKKDKDGKITYVAGKPNGWKVEQGGYPPLAFGFSKGYFYIKANSDRQWLTDKADRNNANPENSEVMKPYSSEYKTSTIAYKMPFAQFPKDCWITFDVAIDWTKYGKEANTILKPGKLDVMMTYTKNKKPQKAHIVNQQEILIGRNDDDGYYFKFGIYRVGNSTVPVTYNLSGYSETAR

[0051] In the present application, when the fusion protein is MBP-F6-HepI, the amino acid sequence it has is as shown in SEQ ID NO:15.

[0052] SEQ ID NO:15:

[0053] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAK AGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSQQKKSGNIPYRVNVQADSAKQSEIIDNKWVAVGINKPYALQYDDKLRFNGKPSYRFELKAEDNSLEGYAAGETKGRIELSYSYATTNDFKKFPPSVYQNAQKLKTVYHYGKGICEQGSSRSYTFSVYIPSSFPDNATTIFAQWHGAPSRTLVATPEGEIKTLSIEEFLALYDR MIFKKNIAHDKVEKKDKDGKITYVAGKPNGWKVEQGGYPPLAFGFSKGYFYIKANSDRQWLTDKADRNNANPENSEVMKPYSSEYKTSTIAYKMP FAQFPKDCWITFDVAIDWTKYGKEANTILKPGKLDVMMTYTKNKKPQKAHIVNQQEILIGRNDDDGYYFKFGIYRVGNSTVPVTYNLSGYSETAR

[0054] This application provides a vector containing a nucleic acid molecule encoding the aforementioned fusion protein.

[0055] This application does not impose any restrictions on the vector, and the appropriate vector can be selected as needed. For example, the vector can be a plasmid.

[0056] This application provides a genetically engineered bacterium that includes the vector described above.

[0057] The present invention does not impose any restrictions on the genetically engineered bacteria, and those skilled in the art can determine them as needed. For example, the genetically engineered bacteria can be Escherichia coli.

[0058] This application provides a method for constructing and expressing a fusion protein, which involves fusing a linker peptide with a target protein. The constructed fusion protein is obtained by linking two target proteins (such as MBP and HepI) in a certain order using a linker peptide.

[0059] The methods for constructing and expressing fusion proteins are well known to those skilled in the art, and for example, the following steps can be used:

[0060] A fusion gene is constructed by tandemly linking a gene encoding a target protein (such as MBP and HepI) with a gene encoding a linker peptide. The linking method includes: designing appropriate primers and obtaining the fusion gene via polymerase chain reaction (PCR); alternatively, the desired fusion gene can be directly synthesized artificially. The fusion gene is then inserted into the multiple cloning site region of an expression vector to obtain an expression vector containing the fusion gene. This expression vector is then transformed into suitable host cells to obtain transformed host cells. The transformed host cells are cultured, and the expression of the fusion protein is induced using an appropriate method. Finally, the fusion protein is extracted and isolated from the host cells.

[0061] Example 1: Construction of a modified fusion protein of maltose-binding protein (MBP) and heparinase (HepI)

[0062] The modified fusion protein provided in this application is constructed by artificially synthesizing a fusion protein gene. This fusion protein gene contains a nucleotide sequence encoding MBP, a nucleotide sequence encoding a linker peptide, and a nucleotide sequence encoding HepI. The nucleotide sequence encoding MBP is identical to that of MBP-R-HepA (patent CN105753945B); the nucleotide sequence encoding HepI is identical to that of HepI in MBP-R-HepA (patent CN105753945B); the nucleotide sequence encoding linker F1 is shown in SEQ ID NO:6: GGTGGTGGCGGCAGC; the nucleotide sequence encoding linker F2 is shown in SEQ ID NO:7: GGTGGTGGCGGCAGCGGTGGCGGCGGTAGC; the nucleotide sequence encoding linker F3 is shown in SEQ ID NO:8: GGTGGTGGCGGCAGCGGTGGCGGCGGTAGCGGCGGTGGTGGATCC; and the nucleotide sequence encoding linker F4 is shown in SEQ ID NO:9: GGTGGTGGCGGCAGCGGTGGCGGCGGTAGCGGCGGTGGATCCG The nucleotide sequence encoding the linker peptide F6 is shown in SEQ ID NO:10: GGTGGTGGCGGCAGCGGTGGCGGCGGTAGCGGCGGTGGTGGATCCG GTGGCGGTGGTTCTGGTGGTGGTGGCAGCGGTGGTGGCGGCAGC.

[0063] All fusion genes had an NdeI restriction site designed at the N-terminus and an EcoRI restriction site designed at the C-terminus. After double digestion with NdeI and EcoRI, the fusion genes were ligated into the pMAL-c2X (New England Biolabs) vector, which had also been double-digested with NdeI and EcoRI, successfully constructing plasmids pMAL-F1-HepI, pMAL-F2-HepI, pMAL-F3-HepI, pMAL-F4-HepI, and pMAL-F6-HepI, respectively. The plasmids were verified for correctness through restriction enzyme digestion and sequencing. These plasmids were then transformed into *E. coli* strain TB1 to construct engineered strains TB1 / pMAL-F1-HepI, TB1 / pMAL-F2-HepI, TB1 / pMAL-F3-HepI, TB1 / pMAL-F4-HepI, and TB1 / pMAL-F6-HepI.

[0064] The genetically engineered bacteria were cultured using the following method: The liquid culture of the genetically engineered bacteria was carried out using M9YGC medium, the composition of which can be found in the literature (Chen Y, Xing XH, Ye F, Kuang Y, Luo M. Production of MBP–HepA fusion protein in recombinant Escherichia coli by optimization of culture medium. Biochem Eng J. 2007; 34:114–21.).

[0065] Add 100 μg / ml to M9YGC medium -1 Ampicillin, shaker speed 170 rpm. Induction culture method for genetically engineered bacteria: Inoculate a single colony from a solid plate into M9YGC medium, pre-culture at 37℃ for 12-16 h, then transfer the culture to fresh M9YGC medium at a 1% (v / v) inoculation rate, and incubate at 37℃ for approximately 3 h until the bacterial cell concentration OD... 600 When the concentration reached 1.0, IPTG was added to a final concentration of 0.24 mM for induction, and the cells were transferred to a 15°C air bath shaker for further culture for 24 h. After culture, the cells were centrifuged at 10000×g at 4°C for 5 min, and the cells were collected and washed three times with 20 mM Tris-HCl buffer (pH 7.4) containing 200 mM NaCl. Finally, the cells were resuspended in 20 mM Tris-HCl buffer (pH 7.4) containing 200 mM NaCl. The resuspended cells were sonicated at low temperature, centrifuged at 15000×g at 4°C for 30 min, and the supernatant was collected to obtain the crude enzyme solution. Affinity purification was performed at 4°C using an AKTA pure25 protein purification system (Cytiva). The MBPTrap HP column (5 mL; Cytiva) was equilibrated with 25 mL of binding buffer (20 mM Tris-HCl buffer, 200 mM NaCl, pH 7.4), the protein supernatant was loaded onto the MBPTrap HP column, and then the column was washed with 50 mL of binding buffer. The fusion protein was eluted with 25 mL of elution buffer (10 mM maltose, 20 mM Tris-HCl buffer, 200 mM NaCl, pH 7.4). The active peak was collected for further analysis. The protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1-1 and Figure 1-2 The crude enzyme solution contained the target fusion protein, and affinity purification achieved good purification results. Further identification showed that the obtained fusion proteins MBP-F1-HepI, MBP-F2-HepI, MBP-F3-HepI, MBP-F4-HepI and MBP-F6-HepI all had heparinase I activity.

[0066] Example 2: Activity analysis of the modified fusion protein

[0067] Modified fusion proteins MBP-F1-HepI, MBP-F2-HepI, MBP-F3-HepI, MBP-F4-HepI, and MBP-F6-HepI were constructed and expressed using the method described in Example 1 of this application, and the fusion proteins were purified by affinity purification. The fusion protein MBP-F5-HepI, containing the linker peptide F5:(GGGGS)5, was used as a control and expressed and purified together with the fusion proteins. To examine the intrinsic changes in the activity of these six fusion proteins with different linker peptide lengths, their heparinase activity was measured.

[0068] The method for detecting the heparinase activity of the modified fusion protein is as follows: The reaction system is 1 mL, including 5 g / L heparin, 20 mM Tris-HCl buffer (pH 7.0), 3.5 mM CaCl2 and 50 mM NaCl. After adding an appropriate amount of enzyme solution, the mixture is quickly mixed to initiate the reaction. The temperature is 30℃. The change in absorbance at 232 nm within 1 min is measured using a spectrophotometer. Heparin has no absorption at 232 nm, while the absorbance coefficient of the unsaturated uronic acid produced by heparin cleavage catalyzed by heparinase at 232 nm is ε. 232 =3.8mM -1 cm -1 The activity of 1 U heparinase is defined as the amount of enzyme required to cleave and produce 1 μmol of unsaturated uronic acid per minute.

[0069] Protein concentration was determined using bovine serum albumin (BSA) as the standard protein, according to Bradford's standard method. To calculate the specific activity of the fusion protein, gel density scanning was performed on the fusion protein separately to estimate the proportion of the target protein in the total protein.

[0070] The activity of the fusion heparinase I with the linker peptide F5:(GGGGS)5 was set to 100% (21.3 U / mg). The relative values ​​of the specific activities of the six fusion proteins are as follows: Figure 2 As shown.

[0071] As can be seen, when the linker peptide is F6, the specific activity of the fusion protein is 38.0 U / mg, which is 1.8 times higher than that of the fusion protein with linker peptide F5. The fusion protein with linker peptide F1 has the highest specific activity, which is 2.4 times that of the fusion protein with linker peptide F5. The specific activities of the fusion proteins with linker peptides F2 and F3 are 2 times and 1.6 times higher than those with linker peptide F5, respectively. The specific activity of the fusion protein with linker peptide F4 is 78% lower than that of the fusion protein with linker peptide F5.

[0072] In summary, the application of linker peptide F6 significantly improves the specific activity of the modified fusion protein compared to the fusion protein with the corresponding linker peptide F5. Furthermore, it also significantly improves the specific activity (15.4 U / mg) of MBP-HepI containing the original linker peptide NSSSNNNNNNNNNNLGIEGRISEFGS in the prior art.

[0073] Example 3: Thermal stability analysis of the modified fusion protein

[0074] All modified fusion proteins were adjusted to a concentration of 0.6 mg / mL using 20 mM Tris-HCl (pH 7.4) containing 200 mM NaCl. They were incubated at 30°C and 35°C, respectively. Residual activity was analyzed at appropriate time intervals. The results are shown below. Figure 3-1 and Figure 3-2 As shown in the figure, the modified fusion protein MBP-F6-HepI with F6 as the linker peptide exhibits significantly superior thermal stability compared to other fusion proteins, regardless of whether the temperature is 30℃ or 35℃.

[0075] Based on the time curves of residual activity of the fusion proteins, the half-lives of different fusion proteins at 30℃ and 35℃ were calculated, and the results are shown in Table 1.

[0076] Table 1. Half-life of fusion heparinases with different linker peptides at 30°C and 35°C.

[0077]

[0078] Combined with Table 1 and Figure 3-1 , Figure 3-2 It can be seen that the thermal stability of MBP-F2-HepI is slightly improved compared to MBP-F5-HepI, while the thermal stability of MBP-F3-HepI and MBP-F4-HepI is close to that of MBP-F5-HepI, with no significant improvement.

[0079] The modified fusion protein MBP-F6-HepI has a half-life of 69 min at 30 °C, which is 1.6 times that of MBP-F5-HepI at the same temperature. Furthermore, its half-life at 35 °C is 13 min, which is 2.2 times that of MBP-F5-HepI at the same temperature. This indicates that the thermal stability of MBP-F6-HepI is significantly improved compared to MBP-F5-HepI.

[0080] Furthermore, the half-life of the modified protein MBP-F6-HepI at 30°C is 6.3 times that of the existing MBP-HepI containing the original linker peptide (11 min), indicating a significant improvement in the thermal stability of MBP-F6-HepI.

[0081] Example 4: Determination of kinetic parameters of the modified fusion protein

[0082] To determine the kinetic parameters of the modified fusion protein, the concentration of heparin was varied in the range of 0-500 μM, and the initial rate of heparin cleavage was measured. The initial reaction rate was fitted with a Michaelis-Menten equation curve, and the kinetic parameters of the modified fusion protein were calculated. The results are shown in Table 2.

[0083] Table 2 Kinetic parameters of fusion heparinases with different linker peptides

[0084]

[0085]

[0086] Note: The values ​​in parentheses in the table are the ratios of the parameter values ​​of the fusion protein to the corresponding parameter values ​​of the MBP-F5-HepI fusion heparinase with linker peptide F5.

[0087] As shown in Table 2, the kinetic analysis of the fusion protein indicates that the catalytic constant k of MBP-F1-HepI is... cat The value is 2.51 times that of MBP-F5-HepI, and the catalytic specificity constant k cat / K m The value is 2.22 times that of MBP-F5-HepI, and the catalytic efficiency is significantly improved.

[0088] K of MBP-F2-HepI and MBP-F3-HepI m The value compared to the K of MBP-F5-HepI m The value decreased slightly, indicating a slight increase in affinity for the substrate. Meanwhile, the catalytic specificity constant k for MBP-F2-HepI and MBP-F3-HepI... cat / K m The values ​​were 2.05 and 1.64 times that of MBP-F5-HepI, respectively, indicating a significant improvement in catalytic efficiency.

[0089] And the K of MBP-F6-HepI m The value compared to the K of MBP-F5-HepI m The value decreased, indicating an increased affinity for the substrate. Additionally, its catalytic specificity constant k... cat / K m The value is 1.89 times that of MBP-F5-HepI, and its catalytic efficiency is significantly improved.

[0090] In summary, regarding maltose-binding protein (MBP) and heparinase I (HepI), compared with the modified fusion protein MBP-F5-HepI constructed using the linker peptides F1, F2, F3, F4, and F6 provided in this application, the modified fusion protein MBP-F5-HepI with F5 as the linker peptide:

[0091] The activity and catalytic efficiency of MBP-F1-HepI are significantly improved, with its activity being 2.4 times that of MBP-F5-HepI and its catalytic efficiency being 2.22 times that of MBP-F5-HepI.

[0092] The activity, thermal stability and catalytic efficiency of MBP-F2-HepI were all improved. Specifically, the activity of MBP-F2-HepI was twice that of MBP-F5-HepI, the half-life of MBP-F2-HepI was slightly longer than that of MBP-F5-HepI, the affinity of MBP-F2-HepI for substrates was slightly increased, and the catalytic efficiency was 2.05 times that of MBP-F5-HepI.

[0093] Both the activity and catalytic efficiency of MBP-F3-HepI were improved. Specifically, the activity of MBP-F3-HepI was 1.6 times that of MBP-F5-HepI. MBP-F3-HepI showed a slight increase in substrate affinity and its catalytic efficiency was 1.64 times that of MBP-F5-HepI.

[0094] MBP-F4-HepI exhibits increased substrate affinity, with catalytic efficiency approaching that of MBP-F5-HepI and thermal stability similar to MBP-F5-HepI, but its activity is somewhat reduced.

[0095] The activity, thermal stability, and catalytic efficiency of MBP-F6-HepI were all improved. Specifically, the activity of MBP-F6-HepI was 1.8 times that of MBP-F5-HepI, and the half-life of MBP-F6-HepI was significantly longer than that of MBP-F5-HepI, being 1.6 times and 2.2 times that of MBP-F5-HepI at 30℃ and 35℃, respectively. MBP-F6-HepI also showed increased affinity for the substrate and its catalytic efficiency was 1.89 times that of MBP-F5-HepI.

[0096] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A modified fusion protein having the amino acid sequence SEQ ID NO:

15.

2. A nucleic acid molecule encoding the fusion protein as described in claim 1.

3. A vector comprising the nucleic acid molecule as described in claim 2.

4. A genetically engineered bacterium comprising the vector as described in claim 3.

5. The application of the fusion protein according to claim 1 in the production of low molecular weight heparin.

6. A method for producing low molecular weight heparin, comprising using a fusion protein as described in claim 1, a nucleic acid molecule as described in claim 2, a vector as described in claim 3, or a genetically engineered bacterium as described in claim 4.

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