WO2021115011A1 - Procédé de préparation de n-acétylgalactosamine transférase - Google Patents

Procédé de préparation de n-acétylgalactosamine transférase Download PDF

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WO2021115011A1
WO2021115011A1 PCT/CN2020/128291 CN2020128291W WO2021115011A1 WO 2021115011 A1 WO2021115011 A1 WO 2021115011A1 CN 2020128291 W CN2020128291 W CN 2020128291W WO 2021115011 A1 WO2021115011 A1 WO 2021115011A1
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protein
ppgalnac
expression
prokaryotic expression
expression vector
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张延�
梁涛
许之珏
贾文娟
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上海交通大学
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

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  • the present invention relates to the field of biotechnology, in particular to a prokaryotic expression system for expressing N-acetylgalactosamine transferase, and further provides preparation by the above-mentioned prokaryotic expression system for expressing N-acetylgalactosamine transferase N-acetylgalactosamine transferase method.
  • Glycosylation modification is an important form of protein post-translational modification. It not only participates in protein shear processing, cell proliferation and differentiation, immune inflammation and other processes, but also has an important impact on recombinant protein drugs. About 70% of recombinant protein drugs have glycosylation modification in their natural state, and N-sugar chains and O-sugar chains are the most common in protein drugs. The lack of glycosylation modification will cause the half-life of these protein drugs in the body to be shortened or the drug effect is reduced.
  • human interferon gamma lacking N-glycosylation modification will be degraded by proteases, resulting in a shortened half-life; ovulation-stimulating drugs Corifollitropin alfa (FSH) Lack of N-glycosylation can lead to heat denaturation and lower potency; O-glycosylation in breast milk protein is beneficial to the health of breastfed infants.
  • FSH Corifollitropin alfa
  • HMOs human milk oligosaccharides
  • glycosyltransferases can be replaced by isoenzymes derived from bacteria or yeast, such as mannosyltransferase Alg1, Alg2, Alg3, Alg9; N-acetylglucosamine transferase ⁇ 3GNT; Sialyltransferase ST3Gal1; Galactosyltransferase B4GalT1.
  • Glycosyltransferases or glycosidases derived from bacteria or other species can be used not only for the synthesis of human N-sugar chains and O-GalNAc sugar chains, but also for the development of glycosylation tool enzymes.
  • ppGalNAc-T enzyme As the isozyme of ppGalNAc-T enzyme has not been found in bacteria or yeast so far, ppGalNAc-T enzyme has become the rate-limiting factor for the rapid synthesis of protein initial O-GalNAc glycosylation in vitro and the large-scale production of O-GalNAc sugar chains. , The development and preparation of ppGalNAc-T enzyme with stable activity and high yield is of great significance to fill the technical defects of protein O-sugar chain synthesis and the subsequent use of enzyme engineering to improve the production of glycosylation tool enzymes. At present, the ppGalNAc-T enzyme is mainly obtained through eukaryotic expression and purification systems, such as human-derived model cells HEK293, insect cells SF9, SF21.
  • Jennifer Lauber et al. first expressed the active ppGalNAc-T enzyme in bacteria.
  • the system uses two co-expression plasmids composed of polycistrons, expressing three molecular chaperones and ppGalNAc-T2 enzyme successively, using EnPresso Medium B produces ppGalNAc-T2 enzyme in Shuffle T7 host strain.
  • the disadvantage of this system is that the expression system is complex and multiple expression plasmids are used; the medium composition is complicated, the raw material is expensive, and the overall yield is low.
  • the purpose of the present invention is to provide a prokaryotic expression system for the expression of N-acetylgalactosamine transferase, and further provide a method for expressing N-acetylgalactosamine transferase.
  • the method for preparing N-acetylgalactosamine transferase by the prokaryotic expression system of the enzyme is used to solve the problems in the prior art.
  • one aspect of the present invention provides a prokaryotic expression vector for expressing N-acetylgalactosamine transferase.
  • the expression vector includes a ppGalNAc-T protein expression cassette and a PDI protein expression cassette.
  • the ppGalNAc-T protein is of human origin.
  • the ppGalNAc-T protein is selected from ppGalNAc-T1 protein, ppGalNAc-T2 protein, ppGalNAc-T3 protein, ppGalNAc-T4 protein, ppGalNAc-T5 protein, ppGalNAc-T6 protein, ppGalNAc-T7 protein , PpGalNAc-T8 protein, ppGalNAc-T9 protein, ppGalNAc-T10 protein, ppGalNAc-T11 protein, ppGalNAc-T12 protein, ppGalNAc-T13 protein, ppGalNAc-T14 protein, ppGalNAc-T15 protein, ppGalNAc-T16 protein, TppGalNAc protein , PpGalNAc-T18 protein, ppGalNAc-T19 protein, ppGalNAc-T20 protein.
  • the amino acid sequence of the ppGalNAc-T protein includes the sequence shown in SEQ ID NO:2.
  • the PDI protein is of human origin.
  • the amino acid sequence of the PDI protein includes the sequence shown in SEQ ID NO:4.
  • the expression vector further includes a Mistic protein expression cassette, and the Mistic is derived from Bacillus subtilis.
  • the amino acid sequence of the Mistic protein includes the sequence shown in SEQ ID NO:6.
  • the ppGalNAc-T protein expression cassette and/or PDI protein expression cassette and/or Mistic protein expression cassette include the same promoter.
  • the expression vector is a multi-gene co-expression vector.
  • the expression vector is constructed by pRSFDuet-1 vector.
  • Another aspect of the present invention provides a prokaryotic expression system for expressing N-acetylgalactosamine transferase, the expression system including the above-mentioned prokaryotic expression vector.
  • the host cell of the prokaryotic expression system is selected from strains with an intracellular oxidizing environment.
  • the host cell of the prokaryotic expression system is selected from Escherichia coli, preferably selected from Rosetta-gami2.
  • Another aspect of the present invention provides a method for preparing N-acetylgalactosamine transferase, which includes the following steps: culturing the prokaryotic expression system described above, thereby expressing N-acetylgalactosamine transferase, and purifying and isolating the N-acetylgalactosamine transferase. -Acetylgalactosamine transferase.
  • Figure 1A shows a schematic diagram of the catalytic form of the ppGalNAc-T enzyme.
  • Figure 1B shows a schematic diagram of structural analysis of human ppGalNAc-T2 protein (RefSeq Accession Number: Q10471).
  • Figure 1C shows a schematic diagram of the structure analysis of human PDI protein (RefSeq Accession Number: P07237).
  • Figure 1D shows a schematic diagram of the plasmid construction strategy of Example 1 of the present invention.
  • Figure 1E shows a schematic diagram of the plasmid construction strategy of Example 1 of the present invention.
  • Figure 2A shows a schematic diagram of gel electrophoresis results after double digestion of pRSFDuet-1 plasmid and double digestion of PDI target fragment.
  • Figure 2B shows a schematic diagram of the gel electrophoresis result of ligating the PDI fragment into the pRSFDuet-1 plasmid after double digestion with the above endonuclease, the Mistic target fragment and the Recombinant ppGalNAc-T2 target fragment.
  • Figure 2C shows a schematic diagram of the gel electrophoresis result of the Recombinant ppGalNAc-T2 target fragment after double digestion.
  • Figure 2D shows a schematic diagram of the gel electrophoresis result of ligating the PDI fragment into the pRSFDuet-1 plasmid after double digestion with the above endonuclease, and the Full Length ppGalNAc-T2 target fragment after double digestion.
  • Figure 2E shows a schematic diagram of the gel electrophoresis result after the connection of the Mistic target fragment and the Full Length ppGalNAc-T2 target fragment.
  • Figure 3A shows a schematic diagram of the result of Coomassie Brilliant Blue staining in Example 2 of the present invention.
  • FIG. 3B shows a schematic diagram of the Western Blot result of Example 2 of the present invention.
  • Figure 4A shows a schematic diagram of the results of Coomassie Brilliant Blue staining and Western Blot in Example 3 of the present invention.
  • 4B shows a schematic diagram of the results of Coomassie Brilliant Blue staining and Western Blot in Example 3 of the present invention.
  • FIG. 5A shows a schematic diagram of the Western Blot result of Example 4 of the present invention.
  • Figure 5B shows a schematic diagram of the results of Coomassie Brilliant Blue staining in Example 4 of the present invention.
  • Fig. 6 is a schematic diagram showing the HPLC spectrum before and after the enzyme activity reaction of the O-glycopeptide of each polypeptide in Example 5 of the present invention.
  • Figure 7 shows a schematic diagram of the mass spectrometry detection results of the Muc5AC and APP polypeptides modified by O-glycosylation in Example 5 of the present invention.
  • Figure 8 is a schematic diagram showing the results of detection of lectin imprints in Example 6 of the present invention.
  • the expression vector and expression system can use a universal medium and pass through After expression and purification, a large number of ppGalNAc-T enzymes with enzymatic activity can be obtained, and subsequent in vitro protein initial O-GalNAc glycosylation and synthesis of O-glyco-modified glycopeptides/glycoproteins can be quickly performed in vitro. On this basis, the present invention has been completed. .
  • the first aspect of the present invention provides a prokaryotic expression vector for expressing N-acetylgalactosamine transferase.
  • the expression vector includes a ppGalNAc-T protein expression cassette and a PDI protein expression cassette.
  • the inventors of the present invention found that the expression of ppGalNAc-T enzyme in a prokaryotic expression system often causes misfolding or expression in inclusion bodies, etc., while in a single prokaryotic expression plasmid of ppGalNAc-T enzyme, human PDI (Protein Disulfide Isomerase (protein disulfide bond isomerase) can help the formation of protein disulfide bond isomerization. It not only helps the protein to fold correctly in E. coli, but also promotes protein solubility, and solves the problem of protein expression in inclusion bodies.
  • the ppGalNAc-T protein is usually of human origin, and preferably may be a recombinant ppGalNAc-T protein, so that it can be suitable for prokaryotic expression system.
  • the ppGalNAc-T protein may be selected from various members of its enzyme family (for example, ppGalNAc-T protein family), for example, the ppGalNAc-T protein may be ppGalNAc-T1 protein, ppGalNAc-T2 protein, ppGalNAc-T3 protein, ppGalNAc-T4 protein, ppGalNAc-T5 protein, ppGalNAc-T6 protein, ppGalNAc-T7 protein, ppGalNAc-T8 protein, ppGalNAc-T9 protein, ppGalNAc-T10 protein, ppGalNAc-T11 protein, ppGalNAc-T12 protein, TppGal ppGalNAc-T14 protein, ppGalNAc-T15 protein, ppGalNAc-T16 protein, ppGalNAc-T17 protein, ppGalNAc-T18 protein, ppGalNAc-T19 protein, or
  • the ppGalNAc-T protein may be ppGalNAc-T2 protein.
  • the amino acid sequence of the ppGalNAc-T protein includes the sequence shown in SEQ ID NO: 2.
  • the nucleic acid coding sequence of the ppGalNAc-T protein includes the sequence shown in SEQ ID NO:1.
  • the PDI protein is of human origin, preferably a recombinant PDI protein, so that it can be applied to a prokaryotic expression system.
  • the amino acid sequence of the PDI protein includes the sequence shown in SEQ ID NO:4.
  • the nucleic acid coding sequence of the PDI protein includes the sequence shown in SEQ ID NO: 3.
  • the expression vector may also include a Mistic protein expression cassette.
  • the inventors of the present invention found that the simultaneous co-expression of Mistic protein in a single prokaryotic expression plasmid of ppGalNAc-T enzyme can improve the solubility of the protein. Specifically, it can help the transmembrane protein to insert into the E. coli cell membrane, thereby achieving protein solubility. Solve the difficulty of protein expression in inclusion bodies.
  • the Mistic protein is usually derived from Bacillus subtilis.
  • the amino acid sequence of the Mistic protein is shown in SEQ ID NO: 6.
  • the coding sequence of the Mistic protein includes the sequence shown in SEQ ID NO: 5.
  • the expression vector is usually a prokaryotic expression vector, more specifically, it can be a bacterial expression vector, preferably an E. coli expression vector.
  • the expression vector is usually a multi-gene co-expression vector, that is, each protein expression cassette (for example, ppGalNAc-T protein expression cassette and/or PDI protein expression cassette and/or Mistic protein expression cassette) can all be located in a single expression vector.
  • the expression vector is constructed by pRSFDuet-1 vector.
  • the ppGalNAc-T protein expression cassette and/or the PDI protein expression cassette and/or the Mistic protein expression cassette may each include a promoter respectively . Multiple protein expression cassettes can also share a promoter.
  • both the ppGalNAc-T protein expression cassette and the PDI protein expression cassette may include a promoter, and ppGalNAc-T may be regulated by the promoters in the ppGalNAc-T protein expression cassette and the PDI protein expression cassette respectively. Protein and PDI protein expression.
  • the expression vector includes a Mistic protein expression cassette and a ppGalNAc-T protein expression cassette that are sequentially connected, and the Mistic protein expression cassette may include a first promoter, so that it can be expressed by Mistic protein.
  • the promoter in the frame simultaneously regulates the expression of the Mistic protein and the ppGalNAc-T protein
  • the PDI protein expression frame may include a second promoter, and the promoter in the PDI protein expression frame may regulate the expression of the PDI protein.
  • the prokaryotic expression vector for expressing N-acetylgalactosamine transferase when multiple promoters are included in the expression vector, these promoters can be the same, which can be induced by a single condition Simultaneous expression of ppGalNAc-T protein and/or PDI protein and/or Mistic protein, for example, the ppGalNAc-T protein expression cassette and/or PDI protein expression cassette and/or Mistic protein expression cassette include the same promoter.
  • the promoter can be T7promoter, Sp6promoter, trp promoter, etc.
  • the second aspect of the present invention provides a prokaryotic expression system for expressing N-acetylgalactosamine transferase, which includes the prokaryotic expression vector provided in the first aspect of the present invention.
  • the host cell of the prokaryotic expression system may generally be a bacterial cell, more specifically an E. coli cell, and a strain with an intracellular oxidizing environment is required.
  • the host cell of the prokaryotic expression system may be Rosetta-gami2.
  • the Rosetta-gami 2 host strain combines the advantages of Rosetta 2 and Origami 2 strains.
  • the intracellular thioredoxin reductase trxB and glutathione reductase gor genes have been mutated.
  • the Rosetta-gami 2 host strain When the heterologous protein is expressed in E. coli , Can alleviate codon preference and enhance the formation of disulfide bonds in the cytoplasm.
  • the Rosetta-gami 2 host strain also carries a chloramphenicol-resistant pRARE2 plasmid, which can provide 7 rare tRNAs and can increase the expression level of proteins containing rare codons.
  • the third aspect of the present invention provides a preparation method of N-acetylgalactosamine transferase, including the following steps: cultivating the prokaryotic expression system provided by the second aspect of the present invention, thereby expressing N-acetylgalactosamine transferase, and purifying The N-acetylgalactosamine transferase is isolated.
  • the preparation method it is necessary to select a suitable medium and culture under conditions suitable for the growth of the host cell.
  • a suitable method such as temperature conversion or chemical induction
  • the N-acetylgalactosamine transferase in the above method can be expressed in the cell, on the cell membrane, or secreted out of the cell.
  • a universal medium can be used to induce expression of the prokaryotic expression system, and the applicable universal medium can be TB medium, LB medium, and the like.
  • IPTG can be used to induce expression in the prokaryotic expression system
  • the time for inducing expression can be 4-24h, 4-8h, 8-12h, or 12-24h
  • the concentration of inducing expression can be 0.01 ⁇ 1mM, 0.01 ⁇ 0.05mM, 0.05 ⁇ 0.1mM, 0.1 ⁇ 0.2mM, 0.2 ⁇ 0.4mM, 0.4 ⁇ 0.6mM, 0.6 ⁇ 0.8mM, or 0.8 ⁇ 1mM.
  • the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art.
  • Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the prokaryotic expression vector and prokaryotic expression system for expressing N-acetylgalactosamine transferase use a single plasmid for co-expression and a host cell with an intracellular oxidizing environment, and express through a conventional general medium.
  • the system and operation method are simple, one-step expression and purification, no subsequent operations such as refolding, and the yield of ppGalNAc-T2 enzyme obtained is better than that of human-derived HEK 293T cell line expressing and purified ppGalNAc-T2 enzyme.
  • the ppGalNAc-T2 enzyme obtained by the expression of this system has good activity. It reacts completely to EA2 polypeptide in 15 minutes, and to Muc5AC polypeptide in 60 minutes. It can react 69.2% to APP-peptide2 in 2 hours. APP-peptide3 can react 24.2% in 2 hours.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel, etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHOD IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, IN Vol.
  • the human ppGalNAc-T2 gene and PDI gene were synthesized in General Biosystems (Anhui) Co., Ltd.;
  • KOD DNA polymerase ligation High ligase, etc. were purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.;
  • DH5 ⁇ competent cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.;
  • Ni-NTA Resin pRSFDuet-1 vector, Rosetta-gaimi2 (pLysS) expression strain were purchased from Millipore;
  • UDP-GalNAc was purchased from Sigma-Aldrich;
  • EA2-FAM polypeptide, Muc5AC-FAM polypeptide, APP-peptide2-FAM polypeptide, APP-peptide3-FAM polypeptide were synthesized in Gill Biochemical Co., Ltd. (Shanghai);
  • the human recombinant P53 protein was expressed in BL21 Escherichia coli, and PCR primer synthesis and sequencing were performed in Shanghai Parsono Biotechnology Co., Ltd.
  • the catalytic form of the ppGalNAc-T enzyme is shown in Figure 1A.
  • the structure of the human ppGalNAc-T2 protein (RefSeq Accession Number: Q10471) was analyzed on the UniProt website ( Figure 1B), and the codons were optimized to make it suitable for E. coli expression.
  • the codon optimization of human ppGalNAc-T2 ie The nucleotide sequence of Recombinant ppGalNAc-T2 (ie The nucleotide sequence of Recombinant ppGalNAc-T2) is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.
  • the structure of the human PDI protein (RefSeq Accession Number: P07237) was analyzed ( Figure 1C), and its N-terminal signal peptide (PDI ⁇ SP, aa:18-508) was truncated, and the codons were optimized to make it suitable for E. coli expression.
  • the optimized nucleotide sequence of the source PDI codon is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.
  • the nucleotide sequence of Bacillus subtilis Mistic is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6; designed according to the multiple cloning site of pRSFDuet-1 and the DNA sequence of ppGalNAc-T2, PDI, and Mistic Primer:
  • Amino acid fragment) upstream primer N-(Amino acid fragment) upstream primer:
  • PDI upstream primer 5’AAAGATATCGATGGATGCACC 3’ (SEQ ID NO: 11)
  • PDI downstream primer 5’CGGGGTACCTTACAGTTCATC 3’ (SEQ ID NO: 12)
  • Plasmid construction was carried out according to the plasmid construction strategy in the attached figure: the synthesized ppGalNAc-T2, PDI and Mistic DNA sequence were used as templates, and the target fragment was amplified by PCR.
  • the PCR reaction conditions were: 94°C for 2min; 94°C for 15s, 52°C for 30s, 68°C2min, 38 cycles; 68°C10min; Mistic target fragments were digested with EcoR I and Nhe I; Full Length ppGalNAc-T2 and Recombinant ppGalNAc-T2 target fragments were digested with BamH I and Sal I; PDI target fragments After EcoR V and Kpn I double digestion, the PDI fragment was ligated into the pRSFDuet-1 plasmid after double digestion with the above endonuclease, and then ligated into the insert protein (human ppGalNAc-T2).
  • the construction strategy is shown in Figure 1
  • the name of the constructed expression plasmid is: pYZL2
  • the name of the specific inserted protein (human ppGalNAc-T2) is:
  • ppGalNAc-T2 human Recombinant ppGalNAc-T2 (hRT2), that is, the above-mentioned Recombinant ppGalNAc-T2 target fragment;
  • Mistic human Recombinant ppGalNAc-T2 (MishRT2), N-terminal to C-terminal includes the above-mentioned Mistic target fragment and the above-mentioned Recombinant ppGalNAc-T2 target fragment connected in sequence;
  • human Full Length ppGalNAc-T2 (hFLT2), that is, the above-mentioned Full Length ppGalNAc-T2 target fragment;
  • Mistic human Full Length ppGalNAc-T2 (MishFLT2)
  • N-terminal to C-terminal includes the above-mentioned Mistic target fragment and the above-mentioned Full Length ppGalNAc-T2 target fragment connected in sequence.
  • Figure 2A is a schematic diagram of the gel electrophoresis results of pRSFDuet-1 plasmid double digestion and PDI target fragment double digestion
  • Figure 2B is the ligation of the PDI fragment into the The gel electrophoresis results of the pRSFDuet-1 plasmid, the Mistic target fragment and the Recombinant ppGalNAc-T2 target fragment after double digestion with the above endonucleases
  • Figure 2C shows the result of double digestion of the Recombinant ppGalNAc-T2 target fragment.
  • FIG. 2D shows the gel electrophoresis results of ligation of the PDI fragment into the pRSFDuet-1 plasmid, Full Length ppGalNAc-T2 after double digestion with the above endonucleases.
  • Figure 2E is a schematic diagram of the gel electrophoresis results of the Mistic target fragment and the Full Length ppGalNAc-T2 target fragment after ligation and double enzyme digestion.
  • Kan + /Cam + /Str + /Tet + kanamycin 50 ⁇ g/ml, chloramphenicol 34 ⁇ g/ml, streptomycin 50 ⁇ g/ml, tetracycline 10 ⁇ g/ ml
  • Kan + /Cam + /Str + /Tet + kanamycin 50 ⁇ g/ml, chloramphenicol 34 ⁇ g/ml, streptomycin 50 ⁇ g/ml, tetracycline 10 ⁇ g/ ml
  • Kan + /Cam + /Str + /Tet + kanamycin 50 ⁇ g/ml, chloramphenicol 34 ⁇ g/ml, streptomycin 50 ⁇ g/ml, tetracycline 10 ⁇ g/ ml
  • Kan + /Cam + /Str + /Tet + kanamycin 50 ⁇ g/ml, chloramphenicol 34 ⁇ g/ml, streptomycin 50 ⁇ g/ml, tetracycline 10 ⁇ g/ ml
  • the cells were collected by centrifugation at 8000g for 5min, 60ml of Lysis Buffer A (25mM Tris-HCl (pH 8.0), 150mM NaCl) was added, the cells were broken at 600Bar for 5min, and centrifuged at 14000g for 30min at 4°C. The precipitate was discarded and the lysed supernatant was collected for protein purification.
  • Lysis Buffer A 25mM Tris-HCl (pH 8.0), 150mM NaCl
  • Wash Buffer 25mM Tris-HCl (pH8.0); 150mM NaCl; 10mM imidazole
  • the ultrafiltration tube (Millipore, 50ml, 30k) was added with 10ml ultrapure water in advance and centrifuged at 4000g for 5min.
  • EA2-FAM polypeptide (SEQ ID NO: 15), Muc5AC-FAM polypeptide (SEQ ID NO: 16), APP-peptide2-FAM polypeptide (SEQ ID NO: 17), APP-peptide3-FAM polypeptide (SEQ ID NO: 18) ) Synthesized in Shanghai Jier Biochemical Co., Ltd. O-glycopeptide enzyme activity reaction system:
  • the ppGalNAc-T enzyme used in the above table was prepared in Example 4, and the reaction system was incubated at 37°C for 30 min.
  • HPLC mobile phase is solution A: H2O+0.05% TFA; solution B: CH3CN+0.05% TFA; HPLC separation conditions: 0-16min, 20%-28%B; 16-18min, 28-80%B; 18- 23min, 80%B; 23-25min, 80%-20%B; 25-30min, 20%B; flow rate: 1ml/min, fluorescence detector excitation wavelength 495nm, emission wavelength 520nm.
  • the HPLC spectra of the O-glycopeptide of each polypeptide before and after the enzyme activity reaction are shown in Figure 6. It can be seen from Figure 6 that compared to the system without the ppGalNAc-T enzyme, the system containing the ppGalNAc-T enzyme has obvious O -Enzymatic reaction of glycopeptides.
  • the human p53 protein (SEQ ID NO: 19) is recombined into the pET28a prokaryotic expression vector, which is a pET28a-p53 prokaryotic expression plasmid, and the human p53 protein has a His tag at its N end.
  • the pET28a-p53 plasmid heat shock method transforms E. coli BL21 competent cells, spreads them on Kan + (kanamycin 50 ⁇ g/ml) plates for selection, and cultures them overnight at 37°C to obtain pET28a-p53 expression strains.
  • the cells were collected by centrifugation at 8000g for 5min, 60ml of Lysis Buffer A (25mM Tris-HCl (pH 8.0), 150mM NaCl) was added, and the cells were broken under high pressure at 600Bar for 5min, and centrifuged at 14000g at 4°C for 30min. The precipitate was discarded and the lysed supernatant was collected for protein purification.
  • Lysis Buffer A 25mM Tris-HCl (pH 8.0), 150mM NaCl
  • the ultrafiltration tube (Millipore, 0.5ml, 10k) was added with 0.5ml ultrapure water in advance and centrifuged at 4000g for 5min. Add the 50-200mM components to the ultrafiltration tube in turn, centrifuge at 14000g at 4°C for 15min; add 0.5ml Lysis Buffer A to dilute the imidazole, centrifuge at 144000g for 15min, repeat more than 10 times (at this time the theoretical concentration of imidazole drops below 2mM). A total of 100 ⁇ l of human recombinant p53 protein was obtained.
  • the ppGalNAc-T enzyme used in the above table was prepared in Example 4, and the reaction system was incubated at 37° C. for 12 h.
  • O-glycoprotein is detected using lectin imprinting.
  • the above reaction system was separated by electrophoresis in 10% SDS-PAGE, and then transferred to a nitrocellulose membrane (NC membrane).
  • NC membrane loaded with O-glycoprotein was blocked in a PBS solution containing 3% BSA for 1 h at room temperature, and then in a clove agglutinin (VVA-HRP, purchased from EY Laboratories) containing 1 ng/ ⁇ l horseradish peroxidase fusion.
  • VVA-HRP clove agglutinin
  • the washed NC membrane reacts with ECL luminescent substrate at room temperature for 1 min.
  • ECL luminescent signal uses AI600 (GE Healthcare) , China) for picture collection, and then the NC membrane loaded with O-glycoprotein was blocked in a TBS solution containing 3% BSA for 1 h at room temperature, and then it was exposed to 1ng/ ⁇ l protein antibody (p53antibody, purchased from Santa Cruz, catalog number -126) TBS solution incubate at room temperature for 1h, and wash 3 times in TBS solution containing 0.1% tween-20.
  • the washed NC membrane is incubated for 1h at room temperature with a secondary antibody containing 0.1ng/ ⁇ l DyLight 680 fusion.
  • the present invention effectively overcomes various shortcomings in the prior art and has a high industrial value.

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Abstract

La divulgation concerne un système d'expression procaryote permettant d'exprimer la N-acétylgalactosamine transférase, et un vecteur d'expression procaryote permettant d'exprimer la N-acétylgalactosamine transférase. Le vecteur d'expression procaryote comprend une cassette d'expression de protéine ppGalNAc-T et une cassette d'expression de protéine PDI. Le vecteur d'expression procaryote et le système d'expression procaryote, permettant d'exprimer la N-acétylgalactosamine transférase, utilisent un plasmide unique de co-expression ainsi qu'une cellule hôte comportant un environnement oxydant intracellulaire, et ils effectuent l'expression par l'intermédiaire d'un milieu de culture général classique. Des opérations de suivi, telles qu'un repli, ne sont pas requises dans le système d'expression procaryote ni dans son procédé de fonctionnement.
PCT/CN2020/128291 2019-12-11 2020-11-12 Procédé de préparation de n-acétylgalactosamine transférase WO2021115011A1 (fr)

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