Method for preparing glycoprotein containing mammalian cell-like N-glycan modified knots by using kluyveromyces marxianus

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for preparing glycoprotein containing a mammalian cell N-glycan modification structure by using kluyveromyces marxianus. Kluyveromyces marxianus FIM1 is used as a starting strain, a gene URA3 on a Kluyveromyces marxianus FIM1 genome is deleted by using a genome editing technology, and then the Kluyveromyces marxianus FIM1 is used as a host strain for recombinant expression of N-glycan glycoprotein; an AT sequence is further inserted into the upstream 5 '-UTR of the gene MNN1, so that the expression of the MNN1 is weakened, and the synthesis of a similar mammalian cell N-glycan sugar chain structure is improved. A carbohydrate chain of N-glycan of the prepared glycoprotein has a carbohydrate chain structure similar to that of mammalian cell N-glycan, hybrid and composite carbohydrate chains exist, and sialic acid and fucose can exist on the carbohydrate chains at the same time. The invention can widely prepare glycoproteins of N-glycans similar to mammalian cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing glycoprotein containing mammalian cell protein N-glycan modified knots by using Kluyveromyces marxianus. Background Art

[0002] N-glycosylation modification after protein translation directly affects the biological function of the protein. The important feature of N-glycan modification of mammalian cell proteins is that they contain both sialic acid (S) and fucose (F) on the N-glycan chain. N-glycans mainly have hybrid or complex sugar chain structures, which can maintain the conformation of N-glycans and ensure the binding of glycoproteins to receptors. At the same time, the sialic acid S at the end of the N-glycan can promote the binding of antibodies to receptors and protect glycoproteins from being removed by non-sialic acid glycoprotein receptors in the liver during blood circulation, while the fucose F located in the core of the sugar chain maintains the stability of the glycoprotein.

[0003] Yeast is a cell factory widely used for recombinant protein expression. Although yeast, like mammalian cells, is a eukaryotic organism and has N-glycosylation modification after protein translation, the natural N-glycosylation modification of yeast proteins is quite different from that of mammalian cells. Early studies found that the Golgi apparatus of Saccharomyces cerevisiae and Pichia pastoris lacks the key enzyme (α-1,2-mannosidase) that processes into hybrid and complex N-glycans, so the N-glycans of yeast proteins cannot form hybrid and complex structures; at the same time, these yeast proteins will extend a polymannosyl chain on the A arm of the N-glycan under the action of α-1,6-mannosyltransferase, and continue to add branches to the trunk under the action of α-1,2 / 3-mannosyltransferase, which not only affects the function of the recombinant protein, but also brings immunogenicity in clinical applications. Sialic acid S and fucose F are sugar components in N-glycans that play important roles in mammalian glycoproteins. There is no report on the simultaneous synthesis of these two important N-glycan components in natural yeast. Therefore, although S.cerevisiae and P.pastoris have been widely used in industrial production, the natural differences in the N-glycosylation modification of their proteins and those in mammalian cells limit the direct application of these natural yeasts to recombinantly express and prepare glycoproteins similar to mammalian cell N-glycan modifications.

[0004] If the defects of the above-mentioned natural yeast in preparing glycoproteins modified with N-glycans similar to mammalian cells can be overcome, and a yeast cell factory can be obtained to manufacture proteins containing N-glycan structures similar to mammalian cells, it will have the advantages of high production efficiency and low production cost, and become the core competitiveness of the industrial biotechnology industry. Kluyveromyces marxianus (K. marxianus) is a new type of yeast with potential for industrial application. It is not only food-grade and safe, but also has the characteristics of fast growth, high temperature resistance, and high secretion capacity. It has been used as a cell factory to efficiently express a variety of recombinant proteins. Kluyveromyces marxianus is naturally present in dairy products, and its long-term coexistence with mammals has naturally formed the ability to modify N-glycans similar to mammalian cells, which is different from the reported modification pathways of N-glycans of Saccharomyces cerevisiae and Pichia pastoris. The present invention provides a method for preparing N-glycan-modified proteins similar to mammalian cells using Kluyveromyces marxianus. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing glycoproteins containing N-glycan modification knots similar to those in mammalian cells.

[0006] The present invention provides a method for preparing a glycoprotein containing an N-glycan modification similar to that of mammalian cells, and uses Kluyveromyces marxianus to prepare a protein modified with N-glycans similar to those of mammalian cells, including weakening the expression of mannosyltransferase MNN1 in Kluyveromyces marxianus cells to promote the synthesis of N-glycans similar to those of mammalian cells; the glycoprotein prepared by using Kluyveromyces marxianus not only has an N-glycan structure similar to that of mammalian cells, but also can contain fucose F and sialic acid S. The present invention can be widely used in the manufacture of proteins containing N-glycan modifications, and these N-glycan modifications directly affect the protein function of the protein, such as functional proteins such as antibodies that require N-glycan modifications to function.

[0007] Specifically, Kluyveromyces marxianus FIM1 is used as a starting strain, and the gene URA3 on the Kluyveromyces marxianus FIM1 genome is deleted by genome editing technology and used as a host strain to recombinantly express N-glycan glycoprotein; the sugar chain of the N-glycan of the prepared glycoprotein has a sugar chain structure similar to that of the N-glycan of mammalian cells; the Kluyveromyces marxianus FIM1 is deposited in the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No.10621.

[0008] Furthermore, based on the knockout of URA3 in Kluyveromyces marxianus FIM1, an AAAAAAAAAAAAAT sequence (denoted as AT sequence) was inserted into the upstream 5'-UTR of the α-1,3-mannosyltransferase gene MNN1 (i.e., Kmarxianus_FIM1_4846) to weaken the expression of the α-1,3-mannosyltransferase gene MNN1 and increase the synthesis of N-glycan sugar chain structures similar to those in mammalian cells.

[0009] The modified structure of N-glycans of the glycoprotein prepared by Kluyveromyces marxianus of the present invention has similar characteristics to the N-glycosylation modification of mammalian cell proteins; specifically:

[0010] The N-glycan structure of the Kluyveromyces marxianus protein includes a hybrid type and a complex type;

[0011] The N-glycan structure of the Kluyveromyces marxianus protein contains both sialic acid and fucose;

[0012] The N-glycan structure processing procedure of the Kluyveromyces marxianus protein is similar to that of mammalian cells.

[0013] The recombinantly expressed N-glycan glycoprotein specifically includes but is not limited to rituximab Fc fragment, ferulic acid esterase, etc.

[0014] More specifically:

[0015] The invention relates to a method for preparing a glycoprotein modified with N-glycans similar to those of mammalian cells, wherein Kluyveromyces marxianus FIM1 is used as a starting bacterium, and the URA3 gene of Kluyveromyces marxianus FIM1 is knocked out by genome editing technology as a host strain; on this basis, an AAAAAAAAAAAAAT sequence is inserted into the 5'-UTR upstream of the gene Kmarxianus_FIM1_4846 by genome editing technology to serve as a host strain for recombinant expression of exogenous proteins; further, a coding gene for an exogenous protein containing an N-glycosylation modification site NXS / T (X is any amino acid except proline) is introduced into the Kluyveromyces marxianus strain, i.e., a gene encoding an exogenous protein containing an N-glycosylation modification site NXS / T's exogenous protein encoding gene is cloned into a recombinant expression vector of Kluyveromyces marxianus, the expression vector comprising a promoter, a secretion signal peptide, a terminator, an autonomous replication sequence pkD1 and a screening marker URA3; the recombinant expression vector containing the exogenous gene is transformed into a ura3Δ defective strain to obtain a positive transformant; after aeration liquid fermentation, the secreted recombinant expressed exogenous protein is obtained in the supernatant of the fermentation liquid; after N-glycopeptide analysis of the secreted recombinant expressed exogenous protein, hybrid and complex N-glycans are present, as well as N-glycans containing sialic acid S and fucose F, and these N-glycan structures are similar to glycoproteins modified by N-glycans of mammalian cells, which are the fundamental characteristics of N-glycan glycoproteins of mammalian cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The expression level of α-1,3-mannosyltransferase gene MNN1 was reduced by inserting the sequence AAAAAAAAAAAAAT into the 5'-UTR upstream of MNN1; (a) the AT sequence (AAAAAAAAAAAAAT) was inserted into the 163696 position of the 5'-UTR upstream of Kluyveromyces marxianus ChrVII chromosome gene MNN1 (FIM1_4846); (b) the sequence AAAAAAAAAAAAAT was inserted into the 5'-UTR upstream of the MNN1 gene to enlarge the stem loop, increase ΔG, and reduce the gene expression level; (c) after the AT sequence was inserted, the expression level of MNN1 was significantly lower than that before the AT sequence was inserted when the strain was cultured for 48 hours and 72 hours.

[0017] Figure 2 To weaken the expression of gene MNN1 in Kluyveromyces marxianus and promote Kluyveromyces marxianus cells to synthesize glycoprotein N-glycan structures similar to those in mammalian cells.

[0018] Figure 3 The figure shows the N-glycan analysis of rhFc recombinantly expressed by Kluyveromyces marxianus; wherein, (a) is the rhFc in the fermentation supernatant after purification; and (b) is the detected N-glycan of rhFc in the fermentation supernatant.

[0019] Figure 4 The N-glycans of ferulic acid esterase recombinantly expressed in Kluyveromyces marxianus cells; wherein, (a) SDS-PAGE analysis of ferulic acid esterase secreted and expressed in Kluyveromyces marxianus cells; (b) N-glycan modifications on recombinantly expressed ferulic acid esterase.

[0020] Figure 5 Comparison of the main sugar chains of Kluyveromyces marxianus protein N-glycans and mammalian cell protein N-glycans.

[0021] Figure 6 The figure shows the analysis and comparison of fucose F and sialic acid S in the N-glycan chains of Kluyveromyces marxianus proteins and mammalian cell proteins; (a) Sophora japonica type II lectin verifies the presence of α-2,3-linked sialic acid; (b) Dictyosporon fasciatus lectin verifies the presence of α-1,3 / 6-linked fucose; Lane 1 is blank SM culture medium; Lane 2 is Kluyveromyces marxianus proteins grown in SM culture medium for 72 h; Lane 3 is blank YD culture medium; Lane 4 is Kluyveromyces marxianus cell proteins grown in YD culture medium for 72 h; Lane 5 is cell proteins of mammalian cell HK-2. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0023] Example 1, knocking out the URA3 gene in Kluyveromyces marxianus, inserting an AT sequence (AAAAAAAAAAAAAT) in the 5'-UTR upstream of the MNN1 gene, reducing the expression level of MNN1, reducing the processing of high mannose chains in Kluyveromyces marxianus, and promoting the synthesis of N-glycan chains similar to those in mammalian cells.

[0024] The URA3 gene is a gene in the uracil synthesis pathway in Kluyveromyces marxianus. If the URA3 gene is knocked out, Kluyveromyces marxianus will lose the ability to synthesize uracil and cannot grow on a culture medium without uracil. The URA3 gene is often used as a marker gene for nutritional deficiency screening and as a screening marker in the construction of yeast genetic engineering strains.

[0025] The MNN1 gene is an α-1,3-mannosyltransferase with the NCBI system number Kmarxianus_FIM1_4846. It is responsible for further forming a branched structure based on the extension of the polymannose chain trunk on the A arm of the N-glycan. It is a high-mannose structure of N-glycans unique to Saccharomyces cerevisiae and Pichia pastoris. The presence of this high mannose will directly affect the function of glycoproteins.

[0026] The method for promoting the synthesis of N-glycan structures similar to mammalian cells provided by the present invention is to introduce an AAAAAAAAAAAAAT sequence ( Figure 1 A), the strain after the insertion of the AT sequence was recorded as ura3ΔFIM1_ChrVII 163696::AT. The addition of the AT sequence strengthened the hairpin structure, increased the ΔG of MNN1 gene transcription, and reduced the expression of the MNN1 gene ( Figure 1 B). The strains before and after the AT sequence was inserted were cultured in shake flasks for 72 hours, and the expression levels of MNN1 at 48 hours and 72 hours were detected by qPCR. The insertion of the AAAAAAAAAAAAAT sequence into the 5'-UTR of the MNN1 gene reduced the relative expression level of MNN1 by about 2.5 times at 48 hours and by about 7 times at 72 hours ( Figure 1 C) The decrease in MNN1 expression level indicates a decrease in the synthesis of polymannose chain branches on the A arm of N-glycans in K. marxianus and an increase in the synthesis of complex and hybrid N-glycans similar to mammalian cells.

[0027] We further tested the changes in the N-glycan structure before and after the insertion of the AT sequence into the 5'-UTR of MNN1 ( Figure 2 ). The analysis of the N-glycan structure of Kluyveromyces marxianus glycoprotein was conducted at Hanno Biotech. The Kluyveromyces marxianus cell proteins were extracted, reduced alkylated and digested with trypsin, desalted by C18 and enriched by ZIC-HILIC, and analyzed by high-resolution RPLC-MS / MS, and matched with the GPseeker database for identification and analysis. After the expression of MNN1 was weakened, 14 new N-glycan structure types were added. These newly added N-glycans all exist in mammalian cells, including complex types 8, 9, 10, 11, 13, and 14; hybrid sugar chains 1, 2, 3, 4, 5, 6, 7, and 12; N-glycan chains containing both fucose and sialic acid are 3, 4, 6, and 10, and sugar chains containing both fucose F and sialic acid S are already recognized as N-glycan features of mammalian cell glycoproteins.

[0028] The present invention uses the well-known genome editing technology to knock out the URA3 gene in Kluyveromyces marxianus. Specifically, the guide RNA of the URA3 gene is inserted into LHZ997 to construct a CRISPR plasmid for knocking out the URA3 gene; nucleotide fragments of about 500 bp in length upstream and downstream of the URA3 gene coding sequence are amplified by PCR, and then the nucleotide fragments of about 500 bp in length upstream and downstream of the URA3 gene are amplified by overlap extension PCR to fuse and amplify the homologous fragments; the CRISPR plasmid and the homologous fragments are co-transformed into Kluyveromyces marxianus by chemical transformation and coated on SD plates. After static culture at 30°C for 2-3 days, single clones are picked on the plate to identify positive clones of URA3 gene knockout by PCR.

[0029] The present invention still adopts the well-known genome editing technology to insert the sequence AAAAAAAAAAAAAT into the upstream of the gene MNN1 (the gene number of NCBI is FIM1_4846), that is, the 163696 position of ChrVII of the Kluyveromyces marxianus chromosome ( Figure 1 A). Insert sequence AAAAAAAAAAAAAT upstream of MNN1 gene. The specific operation is as follows: insert guide RNA near position 163696 upstream of MNN1 gene into LHZ997 for constructing CRISPR plasmid; amplify homologous fragment of AAAAAAAAAAAAAT inserted at position 163696 by PCR; co-transform CRISPR plasmid and homologous fragment into Kluyveromyces marxianus by chemical transformation and spread on SD plate, culture at 30℃ for 2-3 days, pick single clone on the plate and identify positive clone of URA3 gene knockout by PCR.

[0030] Example 2: Antibody Fc protein is prepared using Kluyveromyces marxianus, which contains N-glycan structure similar to mammalian cells.

[0031] In this embodiment, the present invention selects the antibody protein, i.e., the Fc of the monoclonal antibody, to be recombinantly expressed in Kluyveromyces marxianus. The amino acid sequence of Fc contains 1 NXS / T site, which is a recognized modification site of N-glycans and is recognized as a glycoprotein. At the same time, the sugar chain structure of N-glycans directly affects the biological function of Fc in cells. For example, the presence of sialic acid S modification on the sugar chain of N-glycans can promote the binding force of antibodies to receptors, and protect glycoproteins from being removed by non-sialic acid glycoprotein receptors in the liver in the blood circulation. The presence of core fucose F maintains the stability of glycoproteins, and the presence of galactosyl helps to improve the CDC effect of antibodies. The preparation of the Fc of rituximab provided by the present invention in Kluyveromyces marxianus cells is as follows: the coding gene (SEQ ID No.1) of the Fc fragment of rituximab is fully synthesized at a gene synthesis company; it is then cloned into the secretory expression vector of Kluyveromyces marxianus. The expression vector contains the promoter, secretion signal peptide, and terminator sequence required for the recombinant expression of rhFc, and also contains the high-copy sequence pKD1 that replicates autonomously in Kluyveromyces marxianus, and the gene sequence encoding the screening marker URA3. The promoter can be INU1, TEF, or ADH1 promoter, of which the INU1 promoter is preferred; the secretion signal peptide is INU1, α-factor, or SUC2 secretion signal peptide, of which INU1 is preferred. The recombinant plasmid containing the rhFc fragment is transformed into Kluyveromyces marxianus ura3Δ, and the more preferred strain is ura3ΔFIM1ChrVII163696::AT. After screening, positive clones expressing recombinant rhFc are obtained. The single clone is inoculated into the seed solution for overnight culture for 12-16 hours, and is transferred to a 5L fermenter containing 1.5L of base material at an inoculum size of 10%. After 3-4 days of culture, the fermentation broth is collected and centrifuged to obtain the fermentation broth supernatant, which is then separated and purified ( Figure 3 a). The purified protein was sent to Hanno Biotech for N-glycopeptide analysis. From the N-glycans of rhFc obtained in the actual test, the secreted rhFc had a heterozygous type ( Figure 3 b, structure 1) and composite type ( Figure 3 b, structures 2 and 3) N-glycans; fucose F and sialic acid S were detected simultaneously in the N-glycans of secreted rhFc ( Figure 3 b, Structure 3), the presence of galactose L was detected ( Figure 3 b, Structures 1, 2, and 3). These structures are similar to the N-glycan structures in mammalian cells.

[0032] Example 3: Using Kluyveromyces marxianus to prepare N-glycans of biological enzymes, which contain sialic acid and fucose.

[0033] In this embodiment, a biological enzyme protein, namely feruloyl esterase, is selected for recombinant expression in Kluyveromyces marxianus. The amino acid sequence of feruloyl esterase contains 1 NXS / T site, which is a recognized N-glycan modification site. The preparation of feruloyl esterase provided by the present invention in Kluyveromyces marxianus cells is as follows: the feruloyl esterase encoding gene (SEQ ID No.2) is fully gene synthesized in a gene synthesis company; then cloned into the secretion expression vector of Kluyveromyces marxianus. The expression vector contains the promoter INU1, secretion signal peptide INU1, and terminator INU1 sequences required for the recombinant expression of feruloyl esterase, and also contains the autonomously replicated high copy sequence pKD1 in Kluyveromyces marxianus, and the selection marker URA3 encoding gene sequence. The recombinant plasmid containing the feruloyl esterase gene is transformed into the Kluyveromyces marxianus ura3Δ strain, ura3ΔFim1ChrVII163696::AT. After screening, a positive clone of recombinantly expressed feruloyl esterase is obtained. The positive clones were inoculated into YD shake flasks and cultured for 72 h. The fermentation broth was collected and centrifuged to obtain the supernatant of the fermentation broth and then subjected to SDS-PAGE detection ( Figure 4 a), after staining and decolorization, the gel block corresponding to the ferulic acid esterase position on SDS-PAGE was cut off for glycosidase cleavage of sugar chains, and then purified by C18 desalting and detected by mass spectrometry. The N-glycan modification of the secreted ferulic acid esterase is complex, with sialic acid at the end of the sugar chain. One of the complex sugar chains also contains fucose ( Figure 4 b) These structures are also similar to the N-glycan structures in mammalian cells.

[0034] Example 4: Structural comparison of Kluyveromyces marxianus glycoprotein N-glycans and mammalian cell N-glycans.

[0035] The present invention further provides a comparison between the N-glycan structure of Kluyveromyces marxianus glycoprotein and the N-glycan structure of mammalian cells. By analyzing the N-glycan structure, the N-glycan processing process of Kluyveromyces marxianus is provided, showing the N-glycan processing process of Kluyveromyces marxianus similar to that of mammalian cells, further supporting the creativity of Kluyveromyces marxianus in manufacturing N-glycan structures similar to those of mammalian cells. This is currently the only natural yeast cell factory that can process N-glycan structures similar to those of mammalian cells and can also process N-glycans containing fucose F and sialic acid S. The specific description is as follows:

[0036] The present invention uses high-resolution RPLC-MS / MS and GPseeker combined analysis methods at Hanno Biotech to analyze the N-glycopeptide group of Kluyveromyces marxianus protein, and compares it with the N-glycopeptide results of mammalian cell LO2 obtained by the same analysis method. The present invention measured the N-glycans of Kluyveromyces marxianus, which have hybrid and complex structures, and are similar to the N-glycan structures in mammalian cells.

[0037] Among the main sugar chains of N-glycans (or precursor sugar chains of the final structure of N-glycans) measured, 29 main sugar chain structures common to Kluyveromyces marxianus and mammalian cells were detected ( Figure 5 , solid stars and empty stars), and 12 species unique to Kluyveromyces marxianus ( Figure 5 only real stars) and 9 species unique to mammalian cells ( Figure 5 , only empty stars) main sugar chain structure. According to the measured main sugar chain structure, the present invention reconstructs the processing process of the main sugar chain to better compare the similarities and differences between the structures of N-glycans in Kluyveromyces marxianus and mammalian cells. The modification and processing of N-glycans after protein translation all start with tetradecans linked to dolichol. After the endoplasmic reticulum assembly is completed, the tetradecans will be transferred from dolichol to the N site of NXS / T of the nascent peptide chain of the target protein under the action of the OST complex, and then the glucose and mannose residues will be cut in sequence under the action of glycosidase, and the N-acetylglucosamine will be transferred to the mannose residue under the action of the transferase of the Golgi apparatus. The number of transfers and the linking mode are different, forming different main sugar chain structures of N-glycans. On the basis of the main sugar chain of N-glycans, galactose, fucose and / or sialic acid are further added to form the sugar chain structure of mature N-glycans. To this end, this example further uses the biochemical method of lectin imprinting to compare and analyze the fucose F and sialic acid S ( Figure 6 ). The comparative analysis of this example further illustrates that the measured N-glycans of Kluyveromyces marxianus have hybrid and complex structures, and N-glycans containing F and S exist at the same time, which is similar to the N-glycan structure in mammalian cells.

[0038] The examples involve materials and methods.

[0039] Cultivation of Kluyveromyces marxianus.

[0040] The medium used for the basic growth of K. marxianus was YPD medium: 2% polypeptone, 2% glucose, 1% yeast extract, and the culture conditions were 30°C and 220rpm. The recombinant expression strain was cultured in a shake flask using YD medium: 2% yeast extract, 4% glucose, and the culture conditions were 30°C and 220rpm for 72h to detect the expression of foreign proteins in K. marxianus. SD solid medium for transformation coating: 6.7g / L yeast nitrogen base without amino acids, 20g / L glucose, and 20g / L agar powder.

[0041] Plasmid transformation into Kluyveromyces marxianus strains.

[0042] Pick bacteria into 3mL YPD test tube, and culture at 30℃ shaking table for 18-19h; take 1mL bacterial solution, centrifuge at 8000rpm, remove supernatant, wash with 1mL sterile water, centrifuge at 8000rpm, remove supernatant; wash with 1mL 1×TE / LiAc (0.1M / L LiAc, 10mM / L Tris, 1mM / L EDTA), centrifuge at 8000rpm, remove supernatant, repeat once; add 10μL of ligation product (or 3-4μL of plasmid), 600μL PEG solution (0.1M / L LiAc, 10mM / LTris, 1mM / EDTA, 40% PEG 4000) and DTT (final concentration is 10mmol); water bath at 30℃ for 15min, culture at 47℃ for 15min; centrifuge briefly, remove supernatant, add 100μL sterile water, resuspend and apply to plates, and culture in a 30℃ incubator.

[0043] Lectin blotting

[0044] Adjust the cell density of the yeast sample to 30 OD 600 / mL, take an appropriate amount of sample with adjusted cell density, wash with PBS and centrifuge twice to completely remove impurities attached to the cell surface, then resuspend the yeast cells with an equal volume of 200mM NaOH solution and let it stand for 5min, remove NaOH by centrifugation, add an appropriate amount of 5ⅹ Loading Buffer and boil in water for 10min, centrifuge for 5min after cooling to room temperature, take 20μL supernatant for SDS-PAGE, first adjust the voltage to 80V for electrophoresis, adjust the voltage to 120V after the sample enters the separation gel, then transfer the protein band to the PVDF membrane, and use 3% (m / V) BSA (IgG The membrane was blocked with PBST buffer (8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L sodium hydrogen phosphate, 0.24 g / L potassium dihydrogen phosphate, 0.1% (V / V) Tween-20) at room temperature for 1 h on a shaker, and then incubated with biotin-labeled lectin at 4°C overnight. The PVDF membrane was washed with PBST for 5 min and repeated 3 times, and then added ABC-HRP was incubated at 37°C in a shaking incubator for 1-1.5 h, the PVDF membrane was washed with PBST for 5 min and repeated 3 times, the PVDF membrane was covered with developer (Cytiva, catalog number: RPN2232) and photographed.

[0045] The lectin imprint detection protein N-glycan chain containing F and S was obtained by purchasing Biotinylated MaackiaAmurensis Lectin II (Korean Sophora japonica type II lectin, Catalog No.: B-1265-1), Biotinylated AleuriaAurantia Lectin (Dictyosporum lectin, Catalog No.: B-1395-1) and ABC-HRP Kit, Peroxidase (Cat. No.: PK-4000) was used according to the instructions. Maackia Amurensis Lectin II was used to recognize α-2,3-linked S, and Aleuria Aurantia Lectin was used to recognize α-1,3 / 6-linked F.

[0046] Kluyveromyces marxianus fermentation culture.

[0047] The engineered strain is inoculated on YPD solid culture medium and incubated at 30°C for 48 hours for activation. Then a single clone is picked and inoculated into a synthetic culture medium containing glucose, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, vitamins and trace elements, and cultured overnight at 30°C and 220ppm for 14-18 hours. The seed liquid is inoculated into a 5L fermenter containing 1.5L synthetic culture medium at a ratio of 10%. The fermentation process is ventilated and stirred, and the feed is added in a flow-addition manner. The temperature is controlled at 25-35°C, the pH value is controlled between 4.0-6.0 by ammonia water, and the fermentation time is controlled at 48-96 hours. The fermentation process is measured by measuring OD 600 The biomass was monitored and the recombinant proteins were detected by SDS-PAGE.

[0048] Preparation and purification of recombinant proteins by Kluyveromyces marxianus.

[0049] The fermentation supernatant containing the recombinant protein was adjusted to pH 8.0 with NaOH and filtered through a 0.22 μm filter membrane and placed on ice. Add 5 column volumes of deionized water to the gravity column filled with filler to remove ethanol, and then add 5 column volumes of equilibrium buffer (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 10mM imidazole, NaOH to adjust pH to 8.0 and filter through a 0.22 μm filter membrane) to balance the column. Add the sample on ice to the balanced gravity column and keep it for at least 2 minutes to allow the sample to fully contact the medium. Repeat 2-3 times to increase the binding efficiency and collect the flow-through in batches for SDS-PAGE detection. Add 10-15 column volumes of washing solution (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 20mM imidazole, NaOH to adjust pH to 8.0 and filter through a 0.22 μm filter membrane) to the gravity column to remove non-specific binding proteins, and collect the flow-through for SDS-PAGE detection. The protein was eluted with 3-5 column volumes of eluent (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 40-250 mM imidazole, pH adjusted to 8.0 with NaOH and filtered using a 0.22 μm filter membrane), and the flow-through was collected in sections according to the imidazole concentration to obtain the recombinant protein with the highest purity and concentration.

[0050] Structural elucidation of N-glycans from Kluyveromyces marxianus proteins.

[0051] The N-glycopeptide analysis of Kluyveromyces marxianus glycoprotein was commissioned by Hanol (https: / / www.hanol.com.cn / ) to prepare and separate and purify the yeast for detection on the machine in the following way: Yeast was cultured in a shake flask with YD medium (2% yeast extract, 4% glucose) at 30°C, 220rpm, and cultured for 3 days. Yeast cells were obtained by centrifugation, and the cells were washed twice with PBS and then added with cell lysis buffer (4% SDS, 0.1M Tris-HCl, NaOH to adjust pH = 8) and cocktail, placed in an ice bath and ultrasonicated in an ultrasonic cell disruptor, centrifuged at low temperature at 4°C, and the supernatant protein solution or the protein solution after the secretory expression protein was purified was taken. Pre-cooled acetone was added to the protein solution and placed at -20°C overnight. After centrifugation and discarding the supernatant, the protein precipitate was collected and then rinsed with acetone three times. The protein precipitate was re-dissolved with 8M urea solution, the protein concentration was determined, and 1mg of protein was taken, reduced with TCEP at 55°C for 1h, and then IAA was added at room temperature to react in the dark for 30min. The protein solution was taken out from the 1.5 mL centrifuge tube and transferred to a 15 mL centrifuge tube. 20 μg of trypsin was added to the protein solution and the protein was digested overnight on a shaking table. The enzymatically digested peptides were desalted and gradient eluted with C18 and concentrated to dryness under vacuum. The desalted peptides were re-dissolved, and the intact N-glycopeptides were enriched and gradient eluted with ZIC-HILIC, concentrated to dryness under vacuum, and then subjected to LC-MS analysis and library construction search.

[0052] HPLC parameters.

[0053] (1) Liquid chromatography separation parameters

[0054] Analytical column: 360μod×75μid, 75cm long; Packing: Phenomenex Jupiter C18, 5μm,

[0055] Trapped column: 360μod×200μid, 5cm long; Packing: Phenomenex Jupiter C18, 5μm,

[0056] Mobile phase: Buffer A is a mixture of 99.9% H2O and 0.1% FA, and Buffer B is a mixture of 99.9% ACN and 0.1% FA.

[0057] Flow rate: Loading pump mobile phase flow rate (loading): 5μL / min; Nano pump mobile phase flow rate (separation): 300nL / min

[0058] Gradient: Buffer B ratio is 2% within 12 minutes for sample loading; then Buffer B ratio increases linearly from 2% to 40% within 188 minutes for elution; then Buffer B ratio is increased to 95% within 10 minutes, and Buffer B ratio is kept at 95% for 5 minutes for impurity removal; Buffer B ratio is reduced to 2% within 5 minutes, and Buffer B ratio is kept at 2% until the end of the gradient.

[0059] (2) Nano source electrospray ionization parameters

[0060] The temperature of the ion transfer tube was set to 300°C and the spray voltage was 1.9 kV.

[0061] (3) Mass spectrometry and tandem mass spectrometry setting parameters

[0062] Primary mass spectrometry MS: m / z range is 700-2000, mass resolution is 60k (m / z 200), automatic gain control target value (Automatic Gain Control Target, AGC Target) is 3×106, and maximum ion injection time (Maximum Injection Time) is 20 ms.

[0063] Tandem mass spectrometry MS / MS: mass resolution 30k, data-dependent acquisition (DDA) Top 20, automatic gain control target value 5×105, maximum ion injection time 250ms, parent ion isolation window (Isolation Window) 3.0m / z, dynamic exclusion (Dynamic Exclusion) 20.0s, HCD stepped collision normalized energy (steppedNCEs) set to 20%, 30%, 31%.

[0064] GPSeeker database establishment and search process

[0065] Based on the N-glycosylation modification database, trypsin was selected as the protease, the number of missed cleavage sites allowed was set to 1, the static modification was set to alkylation, the MS m / z range was set to 700-2000, and GPSeeker was used to establish the N-glycosylation analysis database, and the mass spectrometry data were matched and identified for complete N-glycopeptides.

[0066] According to the search results, the number of matching peptides (p-MPs) was screened to be ≥5, and the FDR was controlled to be ≤1%. The peptide sequence (p-Seq.), post-translational modification (p-PTMs), and monosaccharide linkage mode (g-Linkage) were deduplicated, and glycopeptides with site and structure scores were screened. Finally, the IDs identification list was obtained, including the number of complete N-glycopeptide identifications (Identification, IDs), peptide sequence (p-Seq.), N-glycosylation site (N-glycosite), N-sugar chain composition (Composition) and linkage mode (g-Linkage), and sugar chain structure diagnostic fragment ions (Structure-diagnostic ions) and other qualitative results.

Claims

1. A method for preparing a glycoprotein containing an N-glycan modification structure similar to that of mammalian cells using Kluyveromyces marxianus, characterized in that: The invention uses Kluyveromyces marxianus FIM1 as a starting strain, uses genome editing technology to delete the gene URA3 on the Kluyveromyces marxianus FIM1 genome and uses it as a host strain to recombinantly express N-glycan glycoprotein; the sugar chain of the N-glycan of the prepared glycoprotein has a sugar chain structure similar to that of the N-glycan of mammalian cells; the Kluyveromyces marxianus FIM1 is deposited in the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No.10621.

2. The method according to claim 1, characterized in that: Based on the knockout of URA3 in Kluyveromyces marxianus FIM1, the AAAAAAAAAAAAAT sequence was inserted into the 5'-UTR of Kmarxianus_FIM1_4846 to weaken the expression of the α-1,3 mannosyltransferase gene MNN1 and increase the synthesis of N-glycan sugar chain structures similar to those in mammalian cells.

3. The method according to claim 2, characterized in that The modified structure of N-glycans of the glycoprotein prepared by Kluyveromyces marxianus has characteristics similar to those of N-glycosylation modification of mammalian cell proteins; Specifically: The N-glycan structure of the Kluyveromyces marxianus protein includes a hybrid type and a complex type; The N-glycan structure of the Kluyveromyces marxianus protein contains both sialic acid and fucose; The N-glycan structure processing procedure of the Kluyveromyces marxianus protein is similar to that of mammalian cells.

4. The method according to claim 3, characterized in that The recombinantly expressed N-glycan glycoproteins include rituximab Fc fragment and ferulic acid esterase.

5. The method according to claim 2, characterized in that: Further, a coding gene of a foreign protein containing an N-glycosylation modification site NXS / T is introduced into the Kluyveromyces marxianus strain, that is, the coding gene of the foreign protein containing the N-glycosylation modification site NXS / T is cloned into a recombinant expression vector of Kluyveromyces marxianus, where X is any amino acid except proline; the expression vector comprises a promoter, a secretion signal peptide, a terminator, an autonomous replication sequence pkD1 and a screening marker URA3; the recombinant expression vector containing the foreign gene is transformed into a ura3Δ defective strain to obtain a positive transformant; after aeration liquid fermentation, the secreted recombinantly expressed foreign protein is obtained in the supernatant of the fermentation liquid; after N-glycopeptide analysis of the secreted recombinantly expressed foreign protein, hybrid and complex N-glycans are present, and N-glycans containing sialic acid S and fucose F are also present; these N-glycan structures are similar to glycoproteins modified by N-glycans of mammalian cells, and are the fundamental characteristics of N-glycan glycoproteins of mammalian cells.

6. Use of Kluyveromyces marxianus in the preparation of glycoproteins containing N-glycan modifications similar to those of mammalian cells.

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