Modified human glycosyltransferase, preparation method and application
阅读说明：本技术 一种改造的人源糖基转移酶、制备方法及应用 (Modified human glycosyltransferase, preparation method and application ) 是由 苏延停 杨秀怡 邓梦雪 李准洁 祝贺 刘武 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种改造的人源糖基转移酶、制备方法及应用。属于生物技术领域。通过去除人源C1GALT1的转移酶膜结合结构域,得到改造蛋白C1GalT1-1。糖结合实验显示,原核表达的C1GalT1-1突变体保持了结合O-GalNAc类型修饰,对其他糖基化修饰并不结合。单糖封闭实验显示,经过GalNAc单糖孵育后,C1GalT1的活性完全被抑制,因此本发明改造蛋白在识别和大规模鉴定O-GalNAc糖基化修饰具有更大的应用潜力。(The invention discloses a modified humanized glycosyltransferase, a preparation method and application thereof. Belongs to the field of biotechnology. The engineered protein C1GalT1-1 was obtained by removing the transferase membrane-binding domain of human C1GALT 1. Carbohydrate binding experiments showed that the prokaryotically expressed C1GalT1-1 mutant retained binding to O-GalNAc type modifications and did not bind to other glycosylation modifications. Monosaccharide blocking experiments show that after incubation of GalNAc monosaccharide, the activity of C1GalT1 is completely inhibited, so that the modified protein has greater application potential in recognition and large-scale identification of O-GalNAc glycosylation modification.)
1. An engineered human glycosyltransferase, wherein an engineered protein C1GalT1-1 is obtained by removing the transferase membrane-binding domain of human C1GalT 1.
2. A method for preparing the engineered human glycosyltransferase of claim 1, comprising the steps of:
1) removing the first 29 amino acid sequences of the N end of the C1GalT1 containing a membrane binding structural domain, wherein the amino acid sequence is shown as SEQ ID NO. 2, and synthesizing a C1GalT1-1 mutant gene with preferred Escherichia coli codon;
2) construction of C1GalT1-1 mutant recombinant plasmid: inserting the C1GalT1-1 mutant gene into Nde1 and EcoR1 restriction enzyme cutting sites of a vector pMal-C5 x;
3) constructing a recombinant expression strain containing the C1GalT1-1 mutant gene: the recombinant plasmid is transformed into a clone bacterium DH5 alpha to extract the plasmid, and finally transformed into an expression bacterium BL21(DE3) to form a BL21-C1GalT1-1 mutant gene;
4) preparation of glycosyltransferase protein C1GalT1-1 mutant: centrifuging 1L of the bacterial liquid at 4000rpm for 20min, removing supernatant, and resuspending and uniformly mixing the precipitate with 40mL of 0.01M PBS; ultrasonic crushing with power of 200W for 10s and stopping for 10s for 90 times; centrifuging at 12000rpm for 20min after crushing, collecting supernatant protein sample, and filtering with 0.45 μm filter membrane to remove insoluble substances; starting to load on the GalNAc-Sepharose 6B affinity chromatography column which is well balanced by PBS in advance, setting the flow rate at 1mL/min, washing the column by PBS buffer after loading, washing off non-specifically bound hybrid proteins until the value detected by the protein detector is not changed, taking about 1h, eluting by GalNAc prepared by 50mM PBS buffer, collecting the elution peak, and using ddH2Performing ultrafiltration, and finally performing freeze-drying to obtain the C1GalT1-1 mutant protein.
3. Use of the engineered human glycosyltransferase of claim 1 to identify an N-acetylgalactosamine-terminated O-linked sugar structure.
Technical Field
The invention belongs to the technical field of biotechnology, and relates to a modified humanized glycosyltransferase, a preparation method and application thereof.
Background
Glycosyltransferases are a large class of enzymes that are widely found in the endoplasmic reticulum and golgi apparatus and are involved in the synthesis of sugar chains in important active substances in the body, such as glycoproteins and glycolipids. The role of this is to transfer the monosaccharide moiety of the corresponding active donor (usually nucleoside diphosphate NDP-sugar) to sugars, proteins, lipids, nucleic acids, etc., which perform the glycosylation process of the latter to perform its biological function. It has now been found that over a hundred glycosyltransferases synthesize over 7000 complex carbohydrate structures, and that most intracellular proteins are modified by glycosylation, and that glycosylation modifications have now been found to be involved in various biological processes and regulate basic physiological activities.
The O-GalNAc (N-acetylgalactosamine) modification is the first modification of most O-linked glycosylation modifications (excluding the O-GlcNAc (N-acetylglucosamine) modification), mainly occurring in the Golgi apparatus, and there are more than 20 glycosyltransferases that can accomplish this, the substrate UDP-GalNAc being covalently linked to a serine or threonine of the protein by the glycosyltransferase ppGalNAc-Ts, first discovered in 1969. Under normal physiological conditions, O-linked glycosylation will generally continue to extend, adding a galactose behind the GalNAc monosaccharide by the galactosyltransferase C1GalT 1. However, C1GalT1 did not perform the glycosylation process alone, and it was necessary to transfer a galactose after the O-GalNAc modification with the help of its specific chaperone Cosmc. The O-GalNAc modification has previously been thought to initiate synthesis in the Golgi apparatus, is mostly secreted extracellularly, and is partly present on the surface of tumor cells, especially some mucin-type glycoproteins. However, recently, it has been reported that a large number of O-GalNAc modified signals were also found in the cell nucleus, and ppGalNAc-T3 glycosyltransferase was successfully identified in the cell nucleus.
The conventional view is that O-GalNAc modification mainly occurs in the secretory pathway, and the main modified target proteins are secretory proteins and membrane proteins. Therefore, the function research of O-GalNAc modification mainly focuses on the functions of membrane proteins and glycoproteins in some body fluids such as serum, because of the close correlation with tumors, and O-GalNAc modification is also called tumor-associated antigen, i.e., Tn antigen. At present, several kinds of Tn antigen positive glycoproteins, such as mucin CA15-3(MUC1) and CA125(MUC16), have been clinically used for the diagnosis and prognosis tracking of various malignant tumors, such as lung cancer, breast cancer, gastric cancer, prostate cancer, ovarian cancer, etc., and the increase of Tn antigen expression generally indicates the increase of tumor malignancy and poor prognosis. In breast cancer, the high expression of Tn antigen can promote tumor cell metastasis by recruiting Galectin-3 and MUC1 to enable tumor cells to attach to endothelial cells. However, it is still unclear what role the Tn antigen plays in tumor development.
There are many tools for identifying O-GalNAc modifications, among which lectins are still widely used for detecting and binding O-GalNAc modifications, such as lectins VVL, TL2, MPL, DBA and Ricin, etc., most of which are derived from plants and fungi. These reports recognize that O-GalNAc modified lectins are non-covalently linked by dimers or tetramers, are not specific, and bind to other monosaccharides such as the terminal sugar structure of galactose. In summary, no lectin specific to O-GalNAc modification exists at present, and the lack of recognition tools also makes the study of O-GalNAc modification difficult.
In addition to lectins, a subject group has found that sugar processing enzymes also have the ability to bind glycoproteins, and that they are used to de-recognize glycosylation modifications by knocking off the catalytically active site of the enzyme, thereby allowing it to retain the ability to bind sugars. For example, the article shows that the modified CpOGA loses the activity of cutting off O-GlcNAc modification but retains the sugar binding activity of binding O-GlcNAc modification, and the modified CpOGA has a good enrichment effect on O-GlcNAc modification in many complex samples. In view of the success of CpOGA engineering, in order to develop a recognition tool for recognizing O-GalNAc modifications, it is necessary to engineer the O-GlcNAc modified extended glycosyltransferase C1GalT1 to screen out a tool for specific binding to O-GalNAc modifications, and to push functional studies on O-GalNAc modifications.
Disclosure of Invention
It is a first object of the present invention to provide an engineered human glycosyltransferase.
The method can be realized by the following technical scheme: an engineered human glycosyltransferase, wherein the engineered protein C1GALT1-1 is obtained by removing the transferase membrane-binding domain of human C1GALT 1.
The second objective of the present invention is to provide a method for preparing an improved human glycosyltransferase, which can be achieved by the following technical scheme:
a preparation method of modified human glycosyltransferase is characterized by comprising the following steps:
1) removing the first 29 amino acid sequences of the N end of the C1GalT1 containing a membrane binding structural domain, wherein the amino acid sequence is shown as SEQ ID NO. 2, and synthesizing a C1GalT1-1 mutant gene with preferred Escherichia coli codon;
2) construction of C1GalT1-1 mutant recombinant plasmid: inserting the C1GalT1-1 mutant gene into Nde1 and EcoR1 restriction enzyme cutting sites of a vector pMal-C5 x;
3) constructing a recombinant expression strain containing the C1GalT1-1 mutant gene: the recombinant plasmid is transformed into a clone bacterium DH5 alpha to extract the plasmid, and finally transformed into an expression bacterium BL21(DE3) to form BL21-C1alT1-1 mutant genes;
4) preparation of glycosyltransferase protein C1GalT1-1 mutant: centrifuging 1L of the bacterial liquid at 4000rpm for 20min, removing supernatant, and resuspending and uniformly mixing the precipitate with 40mL of 0.01M PBS; ultrasonic crushing with power of 200W for 10s and stopping for 10s for 90 times; centrifuging at 12000rpm for 20min after crushing, collecting supernatant protein sample, and filtering with 0.45 μm filter membrane to remove insoluble substances; starting to load on the GalNAc-Sepharose 6B affinity chromatography column which had been equilibrated with PBS in advance, setting the flow rate at 1mL/min, after loading, washing the column with PBS buffer, washing off non-specifically bound foreign proteins until the value detected by the protein detector no longer changes, taking about 1h, finally eluting with 50mM GalNAc (prepared with PBS), collecting the elution peak, and using ddH2Performing ultrafiltration, and finally performing freeze-drying to obtain the C1GalT1-1 mutant protein.
The third purpose of the invention is to provide the use of the modified human glycosyltransferase, namely, the modified human glycosyltransferase is used for identifying a sugar structure taking N-acetylgalactosamine as a terminal.
The invention has the beneficial effects that:
the result of ELISA test on the glycosyltransferase protein C1GalT1-1 mutant provided by the invention shows that C1GalT1-1 can specifically bind to mucin glycoprotein containing O-GalNAc modification and does not bind to other glycoproteins. Therefore, the C1GalT1-1 mutant has great application potential in recognizing O-GalNAc modification.
Drawings
FIG. 1 is an engineered route for engineering C1GalT 1.
FIG. 2 is a SDS-PAGE pattern of the C1GalT1-1 mutant after purification on a GalNAc-coupled column.
FIG. 3 shows the binding results of the C1GalT1-1 mutant to different standard glycoproteins.
FIG. 4 shows the identification of O-GalNAc modification in cells by C1GalT1-1 as a carbohydrate recognition tool.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
The molecular biological methods involved in this experiment are conventional and well known to those skilled in the art. For details not described in the present invention, please refer to the molecular cloning guidelines, edited by Sum Brooks, D.W. Lassel, etc.
[ example 1 ] genetic modification of C1GalT1
According to the structure of C1GalT1 provided by the Uniprot database, in order to increase the soluble expression and not to affect the carbohydrate binding activity of C1GalT1, the modified C1GalT1-1 mutant is obtained by removing the first 29 amino acid sequences from the N-terminus of C1GalT1 containing a membrane binding domain, and the technical strategy is shown in figure one, and all the modified gene sequences are synthesized by the company of Biotechnology engineering (Shanghai).
Example 2 cloning and expression of C1GalT1-1 mutant
Inserting a C1GalT1-1 mutant gene into pMal-C5x through Nde1 and EcoR1 restriction enzyme cutting sites, transforming a recombinant plasmid into a clone bacterium DH5 alpha to extract the plasmid, finally transforming into an expression bacterium BL21(DE3), selecting a single clone to perform overnight amplification culture in 1mL LB culture medium, then transferring the expression bacterium into 10mL of fresh LB culture medium at a ratio of 1:100 to perform culture for 7h, then transferring into 1L of LB culture medium to perform culture for 3h to enable the expression bacterium to grow to a logarithmic phase, namely the OD value reaches 0.6-1.0, and finally performing induced expression for 12h at 20 ℃ by 1mM IPTG.
Example 3 isolation and purification of C1GalT1-1 mutant
1L of the bacterial liquid is centrifuged at 4000rpm for 20min, the supernatant is removed, and 40mL of 0.01MPBS is used for resuspending and mixing the precipitate evenly. Ultrasonic crushing with power of 200W for 10s and stopping for 10s for 90 times. After the disruption, the supernatant was collected by centrifugation at 12000rpm for 20min, and the insoluble matter was removed by filtration through a 0.45 μm filter. The loading was started on a GalNAc-Sepharose 6B affinity column equilibrated in PBS beforehand, the flow rate was set at 1mL/min, after the loading was completed, the column was washed with PBS buffer, and non-specifically bound foreign proteins were washed off until the value detected by the protein detector did not change any more, which required about 1h, and finally elution was carried out with 50mM GalNAc (prepared with PBS), and the elution peak was collected, ultrafiltered with ddH2O, and finally lyophilized. As a result, a large amount of modified C1GalT1-1 mutant protein is obtained. ddH for chromatographic column2Washing with 20% ethanol, and storing.
Example 4 ELISA detection of C1GalT1-1 mutants
Separately diluting Mucin, IgG and Transferin standard glycoprotein to 10ug/mL with coating solution, coating 100 μ L per well overnight at 4 ℃, sucking out the coating solution, washing with 1 ‰ PBST (PBS buffer, 1 ‰ Tween 20) five times, blocking with 5% BSA at room temperature for 1.5h, starting incubating Cgt1 mutant protein solution of 2 μ g/mL, incubating mouse MBP primary antibody (1: 1000) for 1.5h at room temperature, incubating goat anti-mouse secondary antibody (1:5000) for 1h and 1 ‰ PBST washing five times after washing, adding 100 μ L of TMB reaction solution to each well for color development in the dark, stopping the reaction by adding 50 μ L of 1mM hydrochloric acid immediately when blue signal appears, detecting absorbance at 450nm of each well with enzyme labeling instrument, and obtaining the result shown in FIG. 3, wherein the C1GalT1-1 mutant only binds to the protein containing O-Galc modification, no binding to other glycosylation modifications such as N-linked glycosylation at the GlcNAc terminus and the sialic acid terminus.
Example 5 identification of O-GalNAc modification in cells by C1GalT1-1 mutant
Two groups of same HeLa and HepG2 cell complete lysate samples are respectively prepared, samples are prepared simultaneously, 12% SDS-PAGE electrophoresis is carried out, when a 25kD band of a marker approaches to the bottom of a plate, power supply is stopped, membrane transfer is started to a PVDF membrane, then 5% BSA is used for blocking for 1h, one group is incubated with a 2 mu g/mL C1GalT1-1 mutant protein solution at room temperature, the other group is incubated with a mixed solution of 2 mu g/mL C1GalT1-1 mutant protein and 50mM GalNAc which are rotated for 1h in advance at room temperature, after washing, a mouse GST primary antibody (1:2000) is incubated for 1h at room temperature, then a goat anti-mouse secondary antibody (1:5000) marked by horseradish peroxidase is incubated with the mixed solution at room temperature for 1h, after a developing solution reaction, a dark room developing result is shown in a figure 4, the result shows that C1GalT1-1 can be combined with the protein in the cells, and meanwhile Galc modification can be successfully blocked for the combination of C1GalT1-1, these indicate that C1GalT1 binds to a protein in a cell lysate depending on its carbohydrate binding site.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Sequence listing
<110> Hubei science and technology institute
<120> modified humanized glycosyltransferase, preparation method and application
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Gly Ala Leu Ala Pro Pro Ala Val Gly Leu Leu Thr Leu Gly Gly Ala
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Cys Cys Gly Thr Cys Ala Thr Thr Cys Ala Gly Ala Thr Gly Ala Thr
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Ala Ala Cys Gly Gly Thr Cys Ala Gly Ala Ala Cys Cys Ala Thr Cys
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Thr Gly Gly Ala Ala Gly Gly Thr Cys Ala Gly Ala Thr Gly Ala Ala
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Thr Thr Thr Thr Ala Ala Thr Gly Cys Cys Gly Ala Thr Thr Cys Thr
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Ala Ala Ala Ala Cys Ala Cys Cys Gly Ala Thr Ala Thr Cys Gly Cys
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Ala Gly Ala Ala Ala Ala Cys Cys Thr Gly Thr Ala Thr Cys Ala Gly
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Ala Ala Ala Gly Thr Thr Cys Gly Thr Ala Thr Thr Cys Thr Gly Thr
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Cys Cys Cys Gly Cys Ala Gly Ala Ala Thr Cys Thr Gly Gly Ala Ala
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Ala Ala Ala Ala Ala Ala Gly Cys Ala Ala Ala Ala Cys Ala Thr Gly
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Thr Thr Ala Ala Ala Gly Cys Ala Ala Cys Cys Thr Gly Gly Gly Cys
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Gly Cys Ala Thr Thr Thr Cys Ala Gly Thr Ala Thr Gly Thr Thr Cys
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Ala Thr Gly Ala Ala Cys Ala Thr Thr Ala Thr Cys Thr Gly Gly Ala
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Cys Thr Gly Ala Ala Ala Gly Cys Ala Gly Ala Thr Gly Ala Thr Gly
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Ala Thr Ala Cys Cys Thr Ala Thr Gly Thr Thr Ala Thr Thr Cys Thr
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Gly Gly Ala Thr Ala Ala Thr Cys Thr Gly Cys Gly Thr Thr Gly Gly
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Ala Thr Cys Cys Gly Gly Ala Ala Gly Ala Ala Cys Cys Gly Ala Thr
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Thr Thr Thr Ala Ala Ala Cys Cys Gly Thr Ala Thr Gly Thr Thr Ala
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Ala Ala Cys Ala Gly Gly Gly Thr Thr Ala Thr Ala Thr Gly Ala Gly
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Cys Gly Gly Thr Gly Gly Thr Gly Cys Ala Gly Gly Thr Thr Ala Thr
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Gly Cys Ala Gly Cys Ala Gly Cys Ala Thr Thr Gly Ala Ala Gly Ala
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Ala Thr Gly Thr Thr Gly Ala Ala Gly Cys Ala Gly Gly Thr Gly Ala
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Ala Thr Cys Cys Gly Thr Thr Thr Gly Thr Thr Cys Cys Gly Gly Ala
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Cys Cys Thr Thr Thr Thr Gly Gly Thr Ala Thr Thr Gly Gly Ala Ala
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Thr Thr Ala Thr Ala Ala Thr Thr Ala Thr Thr Ala Thr Cys Cys Gly
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Gly Thr Thr Gly Thr Thr Gly Thr Ala Gly Cys Gly Ala Thr Cys Thr
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Gly Gly Cys Ala Gly Thr Thr Ala Gly Cys Thr Thr Thr Cys Ala Thr
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Thr Ala Thr Gly Thr Thr Gly Ala Thr Ala Gly Cys Ala Cys Cys Ala
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Cys Cys Ala Thr Gly Thr Ala Thr Gly Ala Ala Cys Thr Gly Gly Ala
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Ala Thr Ala Thr Cys Thr Gly Gly Thr Thr Thr Ala Thr Cys Ala Thr
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Cys Thr Gly Cys Gly Thr Cys Cys Gly Thr Ala Thr Gly Gly Thr Thr
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Ala Thr Cys Thr Gly Thr Ala Thr Cys Gly Thr Thr Ala Thr Cys Ala
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Gly Cys Cys Gly Ala Cys Cys Cys Thr Gly Cys Cys Gly Gly Ala Ala
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Cys Gly Thr Ala Thr Thr Cys Thr Gly Ala Ala Ala Gly Ala Ala Ala
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Thr Thr Ala Gly Cys Cys Ala Gly Gly Cys Ala Ala Ala Thr Ala Ala
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Ala Ala Ala Thr Gly Ala Ala Gly Ala Thr Ala Cys Cys Ala Ala Ala
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Gly Thr Thr Ala Ala Ala Cys Thr Gly Gly Gly Thr Ala Ala Thr Cys
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Cys Gly
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