Application of an endoglycosidase in hydrolyzing high-mannose N-glycoproteins

Abstract

This invention belongs to the field of biotechnology and relates to the application of an endonuclease in the hydrolysis of high-mannose N-glycoproteins. The endonuclease can recognize and hydrolyze high-mannose N-glycoproteins; the endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1; the hydrolysis temperature is 4–60℃, and the hydrolysis pH is 4.0–10.0. Studies have shown that Endo-LB has the activity of recognizing high-mannose N-glycoproteins, and structurally contains a MucBP domain, exhibiting broad temperature and pH adaptability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of a glycoside endonuclease in the hydrolysis of high-mannose N-glycoprotein. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] β-N-acetylglucosaminidases (ENGases: EC3.2.1.96) are a class of glycosidases that act on N-glycoproteins. They hydrolyze the β-1,4-glycosidic bonds of the core N,N'-diacetylchitobiose in N-glycosylated proteins, generating proteins or polypeptides with a single GlcNAc (referred to as GlcNAc-proteins or GlcNAc-polypeptides, respectively) and releasing the complete oligosaccharide chain. Furthermore, some ENGases also possess transglycosylation activity, enabling the synthesis of N-glycoproteins with various sugar structures. Therefore, ENGases play a crucial role in studying the structure and function of glycoprotein-related N-glycans, as well as in the large-scale preparation of N-glycan chains and glycoprotein modification.

[0004] Currently, ENGases have been widely used in protein N-glycosylation analysis and homogeneous glycoprotein synthesis under various conditions. According to the inventors' research, different ENGase isoenzymes exhibit different preferences for different N-glycan structures and substrate proteins. Therefore, there is a need to find ENGases with different substrate recognition and reaction conditions. Summary of the Invention

[0005] This invention is the first to discover novel ENGases, namely Endo-LB, from the organism Listeria booriae. Studies have shown that Endo-LB has the activity of recognizing high-mannose N-glycoproteins. Structurally, it contains a MucBP (mucin-binding protein) domain and has a wide range of temperature adaptability (4–60°C) and pH adaptability (pH 4.0–10.0).

[0006] Based on the above research results, this invention provides an application of glycoside endonuclease in the hydrolysis of high-mannose N-glycoproteins. Specifically, this invention provides the following technical solution:

[0007] On the one hand, there is the application of an endoglycoside enzyme in the hydrolysis of high-mannose N-glycoproteins, wherein the endoglycoside enzyme can recognize and hydrolyze high-mannose N-glycoproteins; the endoglycoside enzyme is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1; the hydrolysis temperature is 4–60℃, and the hydrolysis pH is 4.0–10.0.

[0008] On the other hand, the application of an endosylase in the analysis of protein N-glycosylation, wherein the endosylase is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1; during the analysis, the temperature is 4 to 60 °C and the pH is 4.0 to 10.0.

[0009] Thirdly, the application of a recombinant Escherichia coli in the hydrolysis of high-mannose N-glycoprotein, wherein the gene expression vector of the recombinant Escherichia coli contains a gene encoding an endonuclease, wherein the endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1.

[0010] Fourthly, the application of a glycoside endonuclease in constructing an N-GlcNAc yeast production platform, wherein the glycoside endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1.

[0011] Fifthly, the application of a recombinant engineered cell in the production of N-GlcNAc modified glycoproteins, wherein the recombinant engineered cell is capable of expressing an endosylase, wherein the endosylase is Endo-LB, the amino acid sequence of which is shown in SEQ ID NO.1, and the endosylase is expressed in the endoplasmic reticulum and / or Golgi apparatus of the recombinant engineered cell.

[0012] The beneficial effects of this invention are as follows:

[0013] Compared with existing ENGases, the Endo-LB provided by this invention has a MucBP (mucin-binding protein) domain that can bind to the host's mucin, and it has a wide range of pH and temperature adaptability. Its high activity under low temperature and low pH conditions makes it more effective in constructing recombinant engineered cells by localizing its expression in host cells such as yeast. Attached Figure Description

[0014] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0015] Figure 1 This is a schematic diagram of the structural domains of Endo-LB in an embodiment of the present invention.

[0016] Figure 2 This is a schematic diagram of the phylogenetic tree of ENGases in the GH18 family in an embodiment of the present invention.

[0017] Figure 3 This is a schematic diagram of partial amino acid sequence alignment of some members of the GH18 family in an embodiment of the present invention.

[0018] Figure 4 This is an SDS-PAGE electrophoresis image of Endo-LB purified in Escherichia coli in an embodiment of the present invention; where M: marker; lane 1: eluted with 10mM imidazole buffer; lanes 2-5: eluted with 250mM imidazole buffer.

[0019] Figure 5 This embodiment of the invention illustrates the hydrolytic activity of purified Endo-LB on glycoproteins with different types of N-glycan chains; wherein, A is an α1-acid substrate protein, B is a human IgG antibody substrate protein, C is a chicken ovalbumin substrate protein, and D is a ribonuclease B (RNase B) substrate protein; red asterisks indicate glycosylated substrates, and blue asterisks indicate deglycosylated substrates.

[0020] Figure 6 This is a graph showing the effect of different pH values ​​on the activity of Endo-LB in the embodiments of the present invention.

[0021] Figure 7 This is a graph showing the effect of different temperatures on the activity of Endo-LB in an embodiment of the present invention.

[0022] Figure 8 This is an SDS-PAGE electrophoresis image of Endo-LB purified with MucBP deficiency in an embodiment of the present invention; where M: marker; lane 1: eluted with 10mM imidazole buffer; lanes 2-4: eluted with 250mM imidazole buffer.

[0023] Figure 9 This is a hydrolytic activity analysis diagram of Endo-LB with MucBP deletion in an embodiment of the present invention; M: marker; ribonuclease B (RNase B) is the substrate protein.

[0024] Figure 10 This is a Western blotting analysis of the deglycosylation efficiency of the RBD region of the SARS-CoV-2 spike protein in engineered yeast strains that express it at the Endo-LB site, as described in this embodiment of the invention; M: marker; the sample is a Western blotting analysis of the expression and purification of the RBD region of the SARS-CoV-2 spike protein in different engineered Pichia pastoris, where WT: wild-type yeast; Endo-T: yeast expressing Endo-T; Endo-LB: yeast expressing Endo-LB. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] Given that different ENGase isoenzymes have different preferences for different N-glycan structures and substrate proteins, this invention proposes the application of a glycosidase in the hydrolysis of high-mannose N-glycoproteins.

[0028] A typical embodiment of the present invention provides the application of an endonuclease in the hydrolysis of high-mannose N-glycoproteins, wherein the endonuclease is capable of recognizing and hydrolyzing high-mannose N-glycoproteins; the endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1; the hydrolysis temperature is 4–60°C, and the hydrolysis pH is 4.0–10.0.

[0029] The hydrolysis temperature can be 4–16℃, 16–25℃, 25–30℃, 30–37℃, 37–42℃, 42–50℃, 50–55℃, 55–60℃, 4–25℃, 16–30℃, 25–37℃, 30–42℃, 37–50℃, 42–55℃, 50–60℃, 4–30℃, 16–37℃, 25–42℃, 30–50℃, 3 Temperatures range from 7 to 55℃, 42 to 60℃, 4 to 37℃, 16 to 42℃, 25 to 50℃, 30 to 55℃, 37 to 60℃, 4 to 42℃, 16 to 50℃, 25 to 55℃, 30 to 60℃, 4 to 50℃, 16 to 55℃, 25 to 60℃, 4 to 55℃, 16 to 60℃, 4℃, 16℃, 25℃, 30℃, 37℃, 42℃, 50℃, 55℃, 60℃, etc.

[0030] The hydrolysis pH can be 4.0–5.0, 5.0–6.0, 6.0–7.0, 7.0–8.0, 8.0–9.0, 9.0–10.0, 4.0–6.0, 5.0–7.0, 6.0–8.0, 7.0–9.0, 8.0–10.0, 4.0–7.0, 5.0–8.0, 6.0–9.0, 7.0–10.0, 4.0–8.0, 5.0–9.0, 6.0–10.0, 4.0–9.0, 5.0–10.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, etc.

[0031] In some embodiments, the high-mannose N-glycoprotein uses ribonuclease B as a substrate. Studies have shown that glycosidases can better recognize and hydrolyze ribonuclease B.

[0032] In some embodiments, the glycoside endonuclease includes a GH18-Endo-S-like domain and a MucBP (mucin-binding protein) domain.

[0033] In another embodiment of the present invention, an application of an endonuclease in the analysis of protein N-glycosylation is provided, wherein the endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1; during the analysis, the temperature is 4-60℃ and the pH is 4.0-10.0.

[0034] During the analysis, the temperature can be 4–16℃, 16–25℃, 25–30℃, 30–37℃, 37–42℃, 42–50℃, 50–55℃, 55–60℃, 4–25℃, 16–30℃, 25–37℃, 30–42℃, 37–50℃, 42–55℃, 50–60℃, 4–30℃, 16–37℃, 25–42℃, 30–50℃, 3 Temperatures range from 7 to 55℃, 42 to 60℃, 4 to 37℃, 16 to 42℃, 25 to 50℃, 30 to 55℃, 37 to 60℃, 4 to 42℃, 16 to 50℃, 25 to 55℃, 30 to 60℃, 4 to 50℃, 16 to 55℃, 25 to 60℃, 4 to 55℃, 16 to 60℃, 4℃, 16℃, 25℃, 30℃, 37℃, 42℃, 50℃, 55℃, 60℃, etc.

[0035] During the analysis, the pH can be 4.0–5.0, 5.0–6.0, 6.0–7.0, 7.0–8.0, 8.0–9.0, 9.0–10.0, 4.0–6.0, 5.0–7.0, 6.0–8.0, 7.0–9.0, 8.0–10.0, 4.0–7.0, 5.0–8.0, 6.0–9.0, 7.0–10.0, 4.0–8.0, 5.0–9.0, 6.0–10.0, 4.0–9.0, 5.0–10.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, etc.

[0036] A third embodiment of the present invention provides the application of recombinant Escherichia coli in the hydrolysis of high-mannose N-glycoprotein, wherein the gene expression vector of the recombinant Escherichia coli contains a gene encoding an endonuclease, and the endonuclease is Endo-LB, the amino acid sequence of which is shown in SEQ ID NO.1.

[0037] In some embodiments, a gene encoding a glycoside endonuclease is recombined into the pET28a vector to construct a gene expression vector, and the constructed gene expression vector is transferred into Escherichia coli to construct recombinant Escherichia coli.

[0038] A fourth embodiment of the present invention provides the application of an endonuclease in constructing an N-GlcNAc yeast production platform, wherein the endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1.

[0039] In some embodiments, a recombinant plasmid is obtained by recombining a primer set expressing a glycosidase into a plasmid, and the recombinant plasmid is introduced into yeast.

[0040] In one or more embodiments, the primer sets are shown as SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0041] In one or more embodiments, the plasmid is pGAPZA.

[0042] In one or more embodiments, the recombinant plasmid is linearized and then introduced into yeast by electroconversion.

[0043] In one or more embodiments, the yeast is Pichia pastoris.

[0044] The fifth embodiment of the present invention provides the application of recombinant engineered cells in the production of N-GlcNAc modified glycoproteins, wherein the recombinant engineered cells are capable of expressing an endonuclease, wherein the endonuclease is Endo-LB, the amino acid sequence of which is shown in SEQ ID NO.1, and the endonuclease is expressed in the endoplasmic reticulum and / or Golgi apparatus of the recombinant engineered cells.

[0045] In some embodiments, the process of constructing the recombinant engineered cells is as follows: the primer set expressing glycosidase is recombined into the plasmid to obtain the recombinant plasmid, and the recombinant plasmid is introduced into yeast.

[0046] In one or more embodiments, the primer sets are shown as SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0047] In one or more embodiments, the plasmid is pGAPZA.

[0048] In one or more embodiments, the recombinant plasmid is linearized and then introduced into yeast by electroconversion.

[0049] In one or more embodiments, the yeast is Pichia pastoris.

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0051] Unless otherwise specified, the materials, reagents, plasmids, strains, etc. used in the following examples were obtained commercially.

[0052] Unless otherwise specified, the experimental methods in the following examples were performed under conditions described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press) or as recommended by the relevant reagent or kit manufacturers.

[0053] Example 1: Analysis of Endo-LB sequences:

[0054] The ENGase in Listeria booriae, named Endo-LB, contains a GH18-EndoS-like domain and a MucBP (mucin-binding protein) domain. Figure 1 No similar ENGases have been reported to date. Its amino acid sequence is shown in SEQ ID NO: 1. A phylogenetic tree was constructed using MEGA6 software to compare Endo-LB with ENGases already reported to belong to the GH18 family. The results show that Endo-LB is more closely related to Endo-BI-1 in the GH18 family of ENGases. Figure 2As shown. Amino acid sequence alignment of Endo-LB with ENGases of the GH18 family revealed that Endo-LB possesses the highly conserved catalytic motif LDXXDXDXE sequence (where X is any amino acid) found in ENGases. However, the overall sequence showed low homology with other known ENGase sequences, with a maximum of 37.6%, indicating that Endo-LB belongs to a newly discovered ENGase of the GH18 family.

[0055] The amino acid sequence of Endo-LB is as follows:

[0056] MRKKSFIAAIIVALCISLVPMFALPSRAVQAEESTNKNFMIYYRAWRDVEMK

[0057] GVNTSLPDENWISMSDIPYGINIVNVFSYVPPGQEELAKPFFDKLKAEYEPEL

[0058] HARGVKLIRGFDYSKLLAIPYAGDFPTEQEFDSYAKALLDELMIPWGLDGL

[0059] DIDMETHPTADEVKISDGVVKALSKYIGPKANNGTLFLYDTNAQNMNPFKN

[0060] VSDSFDLLAYQQYGSGPSRTEMAANAYAPYLPKSQFLPGLTFPEEQDRNRW

[0061] YDTRLPYEDSNIYQVAKHVRENDLAGMFLYAFDRDGKTYNEPDINSVSPSN

[0062] LLWTKTAILEVNGYSVDTAKALAKHHLQRIKYNRNLTSEQVTEVESAIDKA

[0063] TNLYEANVAILGANFETALNPSYDPILEQKLTSIYLTSAFSALDEANAILSKVK

[0064] TNSADIEALKNNKEALENLIGGKKYSESEVNQAVEELLNSIAAVPKPVTVSY

[0065] QDTDGQKLAEDVQLTGKFGGSYEAPQKDIPNYTLQEVKGLERGTFTEIGQS

[0066] VTYVYAKSAPKPAPVVDDNDPVVEEVKAEPAPPVVKNTEDTAKLPQTGDKAASEWPVVVGALAVFASLIVLRRKGL, as shown in SEQ ID NO.1.

[0067] Example 2: Expression and purification of Endo-LB in Escherichia coli:

[0068] 1. Construct recombinant plasmids containing Endo-LB:

[0069] Based on the Endo-LB protein encoding gene sequence (Gene ID: 58719035) published by NCBI, the encoding gene of Endo-LB (Val29-Tyr522, such as SEQ ID NO.1) with the signal peptide removed was synthesized and recombined into the NdeI (CATATG) and XhoI (CTCGAG) multiple cloning sites of the pET28a vector (purchased from Novagen) to construct the pET28a-Endo-LB expression vector.

[0070] 2. Expression of Endo-LB protein:

[0071] 1) Construction of recombinant expression strains:

[0072] The pET28a-Endo-LB plasmid was transformed into E. coli BL21 (E. coli BL21(DE3)pLysS) competent cells, and recombinant E. coli strains containing pET28a-Endo-LB were obtained by screening with kanamycin (final concentration of 50 μg / mL).

[0073] 2) Fermentation of recombinant strains:

[0074] Single colonies of the constructed recombinant strain were picked and placed into 25 mL test tubes containing 5 mL of LB medium. Kanamycin was added to a final concentration of 50 μg / mL, and the tubes were incubated at 37 °C and 200 r / min for 12 h.

[0075] The overnight culture was inoculated into a 1000 mL Erlenmeyer flask containing 500 mL of LB medium at an inoculation rate of 1% (v / v). Kanamycin was added to a final concentration of 50 μg / mL, and the flask was incubated at 37 °C and 200 rpm.

[0076] OD of bacterial culture 600When the concentration of pET28a-Endo-LB was 0.6–0.8, IPTG (Isopropylβ-D-1-thiogalactopyranoside) was added to the recombinant strain containing pET28a-Endo-LB at a final concentration of 0.2 mM, and the strain was cultured at 16 °C and 200 r / min for 16 h to induce expression.

[0077] 3. Extraction of Endo-LB protein:

[0078] Centrifuge the overnight culture at 4°C and 13,000 rpm for 5 minutes, discard all supernatant, and collect the bacterial cells.

[0079] The bacteria were broken down and dissolved using an ultrasonic disruptor, and then centrifuged at 4°C and 13,000 rpm for 10 minutes. The target protein was found in the supernatant.

[0080] The recombinant target protein was purified using a nickel metal ion chromatography column (Ni-NTA).

[0081] a) Column packing: Pour 0.5 mL of Ni-NTA into a 2.5 × 10 cm column and equilibrate the column at 4 °C with 10 column volumes of binding buffer (20 mM Tris-HCl buffer, pH 8.0; 0.3 M sodium chloride; 10 mM imidazole).

[0082] b) Sample loading: Load the lysed supernatant containing the target protein and wash the column with 10 column volumes of washing buffer (20 mM Tris-HCl buffer, pH 8.0; 0.3 M sodium chloride; 20 mM imidazole).

[0083] c) Elution: Elute the target protein with 6 column volumes of elution buffer (20 mM Tris-HCl buffer, pH 8.0; 0.3 M sodium chloride; 250 mM imidazole) and collect the elution buffer containing the Endo-LB protein domain.

[0084] d) Desalting: The protein eluents collected above were ultrafiltered at 4℃ and 4000r / min and desalted with PBS buffer to obtain Endo-LB protein solution.

[0085] 4) Determination of the molecular weight and concentration of Endo-LB protein:

[0086] The Endo-LB protein obtained above was detected by 12% SDS-PAGE electrophoresis.

[0087] Analysis and test results are available in [link to analysis and test results]. Figure 4 .

[0088] The test results showed that Endo-LB protein with a purification degree of >95% was obtained, and the protein concentration was 5 mg / ml.

[0089] Example 3: Determination of Endo-LB deglycosylation activity:

[0090] Ribonuclease B (RNase B), α1-acid, human IgG antibody, and chicken ovalbumin were used as substrates. In a 20 μL reaction system, 0.4 μg Endo-LB and 4 μg of substrate protein were added to pH 7.0 PBS buffer. After mixing, the mixture was incubated at 37°C for 1 h. After the reaction was complete, 5 μL of SDS-PAGE loading buffer was added, and the mixture was boiled at 100°C for 10 min before SDS-PAGE analysis. Under the same conditions, the same amount of PNGase F was used as a positive control.

[0091] Analysis and test results are available in [link to analysis and test results]. Figure 5 .

[0092] The test results showed that Endo-LB can efficiently identify high-mannose glycoprotein substrates, but cannot identify complex and hybrid glycoprotein substrates.

[0093] Example 4: Determination of the optimal pH for the Endo-LB reaction:

[0094] Using the conditions of Example 3, with RNase B as the substrate, 0.4 μg Endo-LB and 4 μg RNase B substrate were added to a 20 μL reaction system at an enzyme-to-substrate ratio of 1:10. The reaction buffers used were citrate-sodium citrate buffer at pH 3, 4, and 5, PBS buffer at pH 6, 7, and 8, and glycine-sodium hydroxide buffer at pH 9 and 10, respectively. After mixing, the reaction was carried out at 37°C for 1 h. After the reaction was completed, 5 μL of SDS-PAGE loading buffer was added, and the mixture was boiled at 100°C for 10 min. Then, SDS-PAGE verification was performed.

[0095] Analysis and test results are available in [link to analysis and test results]. Figure 6 .

[0096] The test results showed that Endo-LB has strong deglycosylation activity in the pH range of 4.0-10.0, and the optimal pH of Endo-LB is pH 6.0.

[0097] Example 5: Determination of the optimal reaction temperature for Endo-LB:

[0098] Using the conditions of Example 3, with RNase B as the substrate, 0.4 μg Endo-LB and 4 μg RNase B were added to a 20 μL reaction system at pH 6.0 at an enzyme-to-substrate concentration ratio of 1:10. After mixing, the mixture was incubated for 1 h at 4°C, 16°C, 25°C, 30°C, 37°C, 42°C, 50°C, 55°C, 60°C, 65°C, and 70°C, respectively. After the reaction was completed, 5 μL of SDS-PAGE loading buffer was added, and the mixture was boiled at 100°C for 10 min, followed by SDS-PAGE verification.

[0099] Analysis and test results are available in [link to analysis and test results]. Figure 7 .

[0100] The test results showed that Endo-LB has strong deglycosylation activity in the range of 4℃ to 60℃, and the optimal reaction temperature of Endo-LB is 37℃.

[0101] Example 6: Construction and activity assay of Endo-LB lacking MucBP:

[0102] 1. Constructing a recombinant plasmid for Endo-LB lacking MucBP:

[0103] Using the encoding gene of Endo-LB (Val29-Tyr522, e.g., SEQ ID NO. 1) synthesized in Example 2 with the signal peptide removed as a template, primers were designed to clone the Endo-LB Truncated (Val29-Val462) expression sequence lacking MucBP and recombined it into the NcoI (CCATTG) and XhoI (CTCGAG) multiple cloning sites of the pET28a vector (purchased from Novagen) to construct the pET28a-Endo-LB Truncated expression vector. The primer sequences are as follows:

[0104] NcoI-Endo-LBt-F:

[0105] CATGCCATGGGCAGCAGCGCGATATTCCGTATGGCATTAAC, as shown in SEQ ID NO.2.

[0106] XhoI-Endo-LBt-R:

[0107] CCGCTCGAGTTAGTGATGATGATGATGATGCACCGGTTTCGGCACCGCCG C, as shown in SEQ ID NO.3.

[0108] 2. Expression and purification of Endo-LB Truncated protein:

[0109] 1) Construction of recombinant expression strains:

[0110] The correctly sequenced pET28a-Endo-LB Truncated plasmid was transformed into E. coli BL21 (E. coli BL21(DE3)pLysS) competent cells, and recombinant E. coli strains containing pET28a-Endo-LBTruncated were obtained by screening with kanamycin (final concentration 50 μg / mL).

[0111] 2) Fermentation of recombinant strains:

[0112] Single colonies of the constructed recombinant strain were picked and placed into 25 mL test tubes containing 5 mL of LB medium. Kanamycin was added to a final concentration of 50 μg / mL, and the tubes were incubated at 37 °C and 200 r / min for 12 h.

[0113] Inoculate the overnight cultured bacterial solution into a 100 mL Erlenmeyer flask containing 500 mL of LB medium at an inoculation rate of 1% (v / v), add kanamycin to a final concentration of 50 μg / mL, and incubate at 37 °C and 200 rpm.

[0114] OD of bacterial culture 600 When the concentration of pET28a-Endo-LB Truncated was 0.6–0.8, IPTG (Isopropylβ-D-1-thiogalactopyranoside) was added to the recombinant strain containing pET28a-Endo-LB Truncated at a final concentration of 0.2 mM, and the strain was cultured at 16 °C and 200 r / min for 16 h to induce expression.

[0115] 3) Extraction of recombinant proteins:

[0116] Centrifuge the overnight culture at 4°C and 13,000 rpm for 5 minutes, discard all supernatant, and collect the bacterial cells.

[0117] The bacteria were broken down and dissolved using an ultrasonic disruptor, and then centrifuged at 4°C and 13,000 rpm for 10 minutes. The target protein was found in the supernatant.

[0118] The recombinant target protein was purified using a nickel metal ion chromatography column (Ni-NTA).

[0119] a) Column packing: Pour 0.5 mL of Ni-NTA into a 2.5 × 10 cm column and equilibrate the column at 4 °C with 10 column volumes of binding buffer (20 mM Tris-HCl buffer, pH 8.0; 0.3 M sodium chloride; 10 mM imidazole).

[0120] b) Sample loading: Load the lysed supernatant containing the target protein and wash the column with 10 column volumes of washing buffer (20 mM Tris-HCl buffer, pH 8.0; 0.3 M sodium chloride; 20 mM imidazole).

[0121] c) Elution: Elute the target protein with 6 column volumes of elution buffer (20 mM Tris-HCl buffer, pH 8.0; 250 mM imidazole; 0.3 M sodium chloride) and collect the elution buffer containing the Endo-LB protein domain.

[0122] d) Desalting: The protein eluents collected above were ultrafiltered at 4℃ and 4000r / min and desalted with PBS buffer to obtain Endo-LB Truncated protein solution.

[0123] 4) Determination of molecular weight and concentration of Endo-LB Truncated protein:

[0124] The Endo-LB Truncated protein obtained above was detected by 15% SDS-PAGE electrophoresis.

[0125] Analysis and test results are available in [link to analysis and test results]. Figure 8 .

[0126] The test results showed that Endo-LB Truncated protein with a purification degree of >95% was obtained, and the protein concentration was 1 mg / ml.

[0127] 5) Deglycosylation activity assay of Endo-LB Truncated protein:

[0128] Based on the deglycosylation results of Endo-LB protein in Example 3, ribonuclease B (RNase B) was used as the substrate. In a 20 μL reaction system with an enzyme-to-substrate ratio of 1:10, 0.4 μg of Endo-LB Truncated and 4 μg of substrate protein were added to PBS buffer at pH 7.0. After mixing, the mixture was incubated at 37°C for 1 h. After the reaction was complete, 5 μL of SDS-PAGE loading buffer was added, and the mixture was boiled at 100°C for 10 min before SDS-PAGE analysis. Under the same conditions, the same amounts of PNGase F, Endo-LB, and Endo-T were used as positive controls.

[0129] Analysis and test results are available in [link to analysis and test results]. Figure 9 .

[0130] The test results showed that the Endo-LB Truncated protein had similar activities to Endo-LB, proving that the MucBP domain does not play an important role in its in vitro deglycosylation activity.

[0131] Example 7: Endo-LB is used to localize the production of N-GlcNAc-modified glycoproteins by Pichia pastoris.

[0132] 1) Construct a recombinant plasmid that localizes and expresses Endo-LB in Pichia pastoris:

[0133] Using the encoding gene of Endo-LB (Val29-Tyr522, e.g., SEQ ID NO. 1) synthesized in Example 2 with the signal peptide removed as a template, primers were designed to clone the Endo-LB expression sequence and recombine it into the XhoI (CTCGAG) and NotI (GCGGCCGC) multiple cloning sites of the pGAPZA vector, which contains the Golgi localization signal and transmembrane region fusion of the Saccharomyces cerevisiae α-1,6-mannosidase (Mnn9) gene, respectively, to construct the pGAPZA-Mnn9-Endo-LB expression vector. The primer sequences used are as follows:

[0134] XhoI-EndoLB-F:

[0135] CCGCTCGAGGTGCAGGCGGAAGAAAGCACCAACAAAAAC, as shown in SEQ ID NO.4.

[0136] NotI-EndoLB-R:

[0137] ATAAGAATGCGGCCGCTTACTTATCGTCATCGTCCTTGTAGTCTAAGCCTT TGCGGCGCAG, as shown in SEQID NO.5.

[0138] 2) Constructing Pichia pastoris engineered strains that specifically express Endo-LB:

[0139] The pGAPZA-Mnn9-Endo-LB recombinant plasmid constructed in step 1) was linearized using AvrII and then introduced into Pichia pastoris by electroporation. Positive clones expressing Endo-LB were screened using different concentrations of Zeocin antibiotic.

[0140] The SARS-CoV-2 spike protein RBD was secreted and expressed in an engineered strain that specifically expressed Endo-LB, and its glycosylation was examined. Wild-type Pichia pastoris and engineered Pichia pastoris strains specifically expressing Endo-T were used as controls.

[0141] Analysis and test results are available in [link to analysis and test results]. Figure 10 .

[0142] The test results showed that the Pichia pastoris engineered strain constructed by localizing Endo-LB expression in Pichia pastoris had better desaccharification efficiency than the previously reported End-T strain.

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a glycosidase in the hydrolysis of high-mannose N-glycoproteins, characterized in that, The glycoside endonuclease can recognize and hydrolyze high-mannose N-glycoproteins; the glycoside endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO. 1; the hydrolysis temperature is 4–60℃, and the hydrolysis pH is 4.0–10.0; The high-mannose N-glycoprotein uses ribonuclease B as a substrate; The glycoside endonuclease includes a GH18-Endo-S-like domain and a MucBP domain.

2. The application of a glycosidase in the analysis of protein N-glycosylation, characterized in that, The glycoside endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.

1. During the analysis, the temperature is 4–60℃ and the pH is 4.0–10.

0. The glycoside endonuclease can recognize and hydrolyze high-mannose N-glycoproteins. The high-mannose N-glycoprotein uses ribonuclease B as a substrate; The glycoside endonuclease includes a GH18-Endo-S-like domain and a MucBP domain.

3. The application of a recombinant *Escherichia coli* in the hydrolysis of high-mannose N-glycoproteins, characterized in that... The gene expression vector of the recombinant Escherichia coli contains a gene encoding an endonuclease, the endonuclease being Endo-LB, whose amino acid sequence is shown in SEQ ID NO.1; The hydrolysis temperature is 4–60℃, and the hydrolysis pH is 4.0–10.

0. The high-mannose N-glycoprotein uses ribonuclease B as a substrate; The glycoside endonuclease includes a GH18-Endo-S-like domain and a MucBP domain.

4. The application of the recombinant *E. coli* as described in claim 3 in the hydrolysis of high-mannose N-glycoproteins, characterized in that... The gene encoding glycoside endonuclease was recombined into the pET28a vector to construct a gene expression vector. The constructed gene expression vector was then transferred into Escherichia coli to construct recombinant Escherichia coli.

5. The application of an endonuclease in constructing an N-GlcNAc yeast production platform, wherein the endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1; the endonuclease can recognize and hydrolyze high-mannose N-glycoproteins. The hydrolysis temperature is 4–60℃, and the hydrolysis pH is 4.0–10.

0. The high-mannose N-glycoprotein uses ribonuclease B as a substrate; The glycoside endonuclease includes a GH18-Endo-S-like domain and a MucBP domain.

6. The application of the glycoside endonuclease as described in claim 5 in constructing an N-GlcNAc yeast production platform, characterized in that, The primer set expressing glycosidase was recombined into the plasmid to obtain the recombinant plasmid, and the recombinant plasmid was introduced into yeast; The primer sets are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; The plasmid is pGAPZA; The recombinant plasmid was linearized and then introduced into yeast by electroporation. The yeast in question is Pichia pastoris.

7. The application of a recombinant engineered cell in the production of N-GlcNAc modified glycoprotein, wherein the recombinant engineered cell is capable of expressing an endosylase, wherein the endosylase is Endo-LB, the amino acid sequence of which is shown in SEQ ID NO.1, and the endosylase is expressed in the endoplasmic reticulum and / or Golgi apparatus of the recombinant engineered cell; The glycoside endonuclease can recognize and hydrolyze high-mannose N-glycoproteins; The hydrolysis temperature is 4–60℃, and the hydrolysis pH is 4.0–10.

0. The high-mannose N-glycoprotein uses ribonuclease B as a substrate; The glycoside endonuclease includes a GH18-Endo-S-like domain and a MucBP domain.

8. The use of the recombinant engineered cells as described in claim 7 in the production of N-GlcNAc modified glycoproteins, characterized in that, The process of constructing the recombinant engineered cells is as follows: the primer set expressing glycoside endonuclease is recombined into the plasmid to obtain the recombinant plasmid, and the recombinant plasmid is introduced into yeast; The primer sets are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; The plasmid is pGAPZA; The recombinant plasmid was linearized and then introduced into yeast by electroporation. The yeast in question is Pichia pastoris.

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