CN111334485A - Prokaryotic expression method of human N-acetylglucosamine transferase II and product thereof - Google Patents
Prokaryotic expression method of human N-acetylglucosamine transferase II and product thereof Download PDFInfo
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Abstract
The invention provides a human N-acetylglucosamine transferase II prokaryotic expression method and a product thereof, relating to the aspects of expression of human GnT-II delta TM in Escherichia coli, purification of GnT-II delta TM, in-vitro activity detection of GnT-II delta TM and the like. The invention successfully expresses and purifies the human GnT-II delta TM in the escherichia coli, and the enzyme has catalytic activity in vitro, solves the technical problems that the prokaryotic expression of the mammalian membrane protein GnT-II is easy to degrade and difficult to purify, and can prepare the GnT-II delta TM in large quantity. The recombinant humanized GnT-II with in vitro activity is obtained, so that the problem of enzymatic synthesis of an important oligosaccharide structure Gn2Man3Gn2 is solved, and the development of sugar chemistry and sugar biology is promoted.
Description
Technical Field
The invention belongs to the technical field of molecular biology and biochemistry, and particularly relates to a prokaryotic expression method of humanized N-acetylglucosamine transferase II and a product thereof.
Background
Glycosylation is the process of modifying proteins or lipids with sugars (oligosaccharides) to form glycocomplexes, and is one of the major forms of post-translational modification in eukaryotic cells. The N-glycosylation modification on the protein directly influences the structure and the function of the protein and has important physiological significance. N-oligosaccharides have various forms, and the biosynthesis pathway thereof is involved in various glycosyltransferases, so that the preparation and property research of glycosyltransferases are one of the important directions in the field of sugar science. N-acetylglucosaminyltransferase II (i.e., GnT-II) is one of the key enzymes in the processing of glycoprotein N-sugar chains in mammalian cells, catalyzing the formation of complex N-sugar chains.
At present, the expression of human N-acetylglucosamine transferase II is successfully realized only in insect cell, animal cell and yeast cell expression systems, wherein the protein expressed by the insect cell and the animal cell is purified, but the yield is low and the cost is high. The expression of a large amount of activity of GnT-II protein in vitro has not been reported yet. Prokaryotic expression systems, such as E.coli, have the advantages of high yield, less interfering proteins and the like, and are ideal systems for expressing recombinant GnT-II proteins in large quantities in vitro. However, no reports of expressing human GnT-II by Escherichia coli have been made so far, and the main reason is that GnT-II is a mammalian membrane protein which is resident by Golgi apparatus, and the GnT-II is easy to degrade in a prokaryotic system, and is difficult to express and purify.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks.
Therefore, in one aspect of the present invention, the present invention overcomes the deficiencies of the prior art and provides a prokaryotic expression method of human N-acetylglucosaminyltransferase II.
In order to solve the technical problems, the invention provides the following technical scheme: a prokaryotic expression method of human N-acetylglucosamine transferase II comprises the following steps,
prokaryotic expression of GnT-II: expressing human GnT-II delta TM in escherichia coli, wherein the GnT-II delta TM is human N-acetylglucosamine transferase II with 1-87 amino acids at the N end cut off;
purification of GnT-II: purifying the prokaryotically expressed GnT-II delta TM;
1-87 amino acids at the N end of GnT-II are cut off to construct a prokaryotic expression vector;
transforming prokaryotic host bacteria, coating on a plate culture medium, and standing overnight;
screening single colonies of host bacteria, inoculating the single colonies to an LB liquid culture medium, and carrying out shaking culture overnight to obtain a bacterial liquid;
inoculating the bacterial liquid to a TB liquid culture medium, carrying out shake culture, and then carrying out cooling culture;
adding an inducer, carrying out shake culture, centrifugally collecting thalli, suspending in a buffer solution, carrying out ultrasonic crushing, centrifuging, and collecting a supernatant;
and (3) washing the affinity chromatography column by using the buffer solution, loading the supernatant, and collecting the eluent to obtain the human N-acetylglucosamine transferase II.
As a preferred embodiment of the prokaryotic expression method of the human N-acetylglucosamine transferase II of the present invention: the GnT-II is GnT-II delta TM, aa 88-443; the prokaryotic expression vector is pET28a-GnT-II delta TM; the transformed prokaryotic host bacterium is ROSETTA.
As a preferred embodiment of the prokaryotic expression method of the human N-acetylglucosamine transferase II of the present invention: the plate culture medium is LB, kanamycin and chloramphenicol plate culture medium; the LB liquid culture medium is LB, kanamycin and chloramphenicol liquid culture medium; the TB liquid culture medium is TB, kanamycin and chloramphenicol; the buffer solution is Tris/HCl and NaCl buffer solution; the inducer is IPTG; the affinity chromatographic column is a nickel ion affinity chromatographic column.
As a preferred embodiment of the prokaryotic expression method of the human N-acetylglucosamine transferase II of the present invention: the buffer solution has the concentration of 25mM of Tris/HCl and 150mM of NaCl respectively.
As a preferred embodiment of the prokaryotic expression method of the human N-acetylglucosamine transferase II of the present invention: the inducer concentration is 0.1 mM.
As a preferred embodiment of the prokaryotic expression method of the human N-acetylglucosamine transferase II of the present invention: the pH of the buffer was 8.0.
As a preferred embodiment of the prokaryotic expression method of the human N-acetylglucosamine transferase II of the present invention: the strain is inoculated in an LB liquid culture medium, and is subjected to shaking culture overnight, wherein the culture temperature is 37 ℃; inoculating the bacterial liquid to a TB liquid culture medium, carrying out shake culture, and then carrying out cooling culture, wherein the temperature is 37 ℃, the oscillation rate is 200r/min, the oscillation time is 3h, the temperature is reduced to 16 ℃, and the cooling culture time is 1 h; adding an inducer, and carrying out shaking culture, wherein the frequency of the shaking culture is 200r/min, and the culture time is 20 h; the thalli is collected by centrifugation, the centrifugation speed is 9000r/min, and the centrifugation time is 60 min.
As a preferred embodiment of the prokaryotic expression method of the human N-acetylglucosamine transferase II of the present invention: inoculating the bacterial liquid to a TB liquid culture medium, carrying out shake culture, and then carrying out cooling culture to obtain bacterial liquid OD6000.6 to 0.8.
As a preferred embodiment of the prokaryotic expression method of the human N-acetylglucosamine transferase II of the present invention: and washing the affinity chromatography column by using the buffer solution, loading the supernatant, and collecting eluent, wherein the volume ratio of the buffer solution to the affinity chromatography column is 10: 1, and the flow rate of the buffer solution is 1 min/mL.
As another aspect of the present invention, the present invention overcomes the disadvantages of the prior art by providing a human N-acetylglucosamine transferase II produced by the prokaryotic expression method of the human N-acetylglucosamine transferase II.
In order to solve the technical problems, the invention provides the following technical scheme: the human N-acetylglucosamine transferase II prepared by the prokaryotic expression method of the human N-acetylglucosamine transferase II is characterized in that: the michaelis constant of the human N-acetylglucosamine transferase II is 55.98 mu M.
The invention has the beneficial effects that: the recombinant human N-acetylglucosamine transferase II (namely GnT-II) is expressed and purified by pronucleus, and the recombinant human N-acetylglucosamine transferase II has activity in vitro, so that the protein can be prepared in a large amount in a short time. The invention has the characteristics of less impurity protein, easy purification, low cost, convenience and the like, solves the technical problems of easy degradation and difficult purification of prokaryotic expression of mammalian membrane protein GnT-II, solves the bottleneck of enzymatic synthesis of oligosaccharide structure Gn2Man3Gn2 by the high activity of GnT-II delta TM, and promotes the development of glycochemistry and glycobiology.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is an immunoblot (western blotting) assay of GnT-II. DELTA.TM proteins (FIG. 1A) 1: cell lysate before induction, 2: post-induction cell lysate; and GnT-II. DELTA. TM. protein electrophoresis SDS-PAGE Coomassie Brilliant blue staining pattern (FIG. 1B) 1: blue Plus II Marker, 2: purified GnT-II. DELTA.TM protein.
FIG. 2 is a reaction process of GnT-II protein in vivo (FIG. 2A) and HPLC detection of purified GnT-II. DELTA. TM protein in vitro activity (FIG. 2B) 1: GnT-II. DELTA. TM and GnT-I. DELTA. TM reaction systems, 2: GnT-I Δ TM reaction system, 3: and (4) a standard sample.
FIG. 3 is a substrate-specific assay of GnT-II protein for two non-natural substrates designed (FIG. 3A); activity assay of GnT-II. DELTA.TM protein for Fmoc-Asn-Gn2Man3 (FIG. 3B), 1: GnT-II Δ TM reaction system, 2: a standard sample; activity assay of GnT-II. DELTA. TM proteins for Fmoc-Asn-Gn2Man3GnGal (FIG. 3C), 1: GnT-II Δ TM and GalT reaction system, 2: GalT reaction system, 3: GnT-I.DELTA.TM reaction system.
FIG. 4 is a Km value fitting graph of GnT-II. DELTA.TM protein.
FIG. 5 is a graph showing the effect of reaction temperature on the in vitro activity of GnT-II. DELTA.TM proteins.
FIG. 6 is a graph showing the effect of pH of the reaction solution on the in vitro activity of GnT-II. DELTA.TM protein.
FIG. 7 is a graph showing the effect of divalent cation type on the in vitro activity of GnT-II. DELTA.TM proteins.
FIG. 8 is a graph showing the effect of divalent manganese ion concentration on the in vitro activity of GnT-II. DELTA.TM proteins.
FIG. 9 shows the results of experiments in which the pesticide-GnT-II was expressed by adding a pesticide tag.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1: prokaryotic expression purification of human GnT-II delta TM
1-87 amino acids at the N end of GnT-II are cut off (GnT-II delta TM, aa 88-443), and a prokaryotic expression vector pET28a-GnT-II delta TM is constructed. The recombinant prokaryotic expression plasmid is transformed into a ROSETTA prokaryotic expression host bacterium, the ROSETTA prokaryotic expression host bacterium is coated on an LB + kanamycin + chloramphenicol (34 mu g/mL) plate, a single colony is picked from the transformed plate the next day, and the single colony is inoculated in 5mL LB + kanamycin + chloramphenicol liquid culture medium and is subjected to shaking culture at 37 ℃ for overnight. Inoculating 2mL of overnight cultured bacterial liquid into 200mL of TB + kanamycin + chloramphenicol liquid culture medium, and performing shake culture at 37 deg.C and 200r/min for 3h to make OD600Reaching 0.6-0.8, then cooling to 16 ℃, continuing to culture for 1h, adding IPTG with the final concentration of 0.1mM, and carrying out induced culture for 20h at 200 r/min. The thalli are collected by centrifugation, resuspended in 20mL buffer solution A (25mM Tris/HCl, 150mM NaCl, pH 8.0), ultrasonically broken pET28a-GnT-II delta TM recombinant bacteria, centrifuged at 8000r/min for 40min, the precipitate is discarded, and the supernatant is collected for purification.
GnT-II. DELTA. TM protein purification: HisTrap HP 1mL affinity chromatography column was first 10 volumes of A flow 1min/mL after equilibration, then 10mL supernatant was applied, and then 10mL each column was washed with 20 or 60 or 200mM imidazole in buffer A, and collected in centrifuge tubes. Samples were run on SDS-PAGE.
FIG. 1A confirms protein expression using anti-His antibody detection, and FIG. 1B shows successful purification of GnT-II. DELTA.TM described above, wherein the protein size of GnT-II. DELTA.TM is about 48 kDa.
Example 2: in vitro Activity assay of GnT-II. DELTA.TM
A standard enzyme activity assay is as follows (50. mu.L): 100mM MES/NaOH (pH 6.0), 10mM MnCl20.2 μ M PA-Gn2Man3, 0.2mM UDP-GlcNAc, 20 μ g/mL GnT-I Δ TM, 200 μ g/mL GnT-II Δ TM, the reaction was incubated at 37 ℃ for more than 5 h. And after the reaction is finished, centrifuging the reaction system for 5min at 15000r/min, taking the supernatant, detecting by high performance liquid chromatography, and comparing the reaction system with a standard sample.
The result is shown in FIG. 2B, and the peak of the product obtained from the reaction system is consistent with the elution time of the PA-Gn2Man3Gn2 standard, which indicates that the GnT-II delta TM obtained from the Escherichia coli expression system has catalytic activity in vitro.
Example 3: mie's constant determination of GnT-II. DELTA.TM
The enzyme reaction system used for the assay was as follows (4. mu.L): 100mM MES/NaOH (pH 6.0), 10mM MnCl21-6 μ M (1.4656 μ M, 2.93 μ M, 6 μ M) Fmoc-Asn-Gn2Man3, 0.2mM UDP-GlcNAc, 20 μ g/mL GnT-I Δ TM, reacted at 37 ℃ for 2 hours, then the reaction system was inactivated at 100 ℃ for 5min, followed by addition of 125 μ g/mL GnT-II Δ TM and reaction at 37 ℃ for 30 min. And after the reaction is finished, centrifuging the reaction system at 15000r/min for 5min, taking supernate, detecting by high performance liquid chromatography, and comparing the reaction system with a standard sample.
The results show that the reaction speed and the substrate concentration can be fitted to a standard straight line calculated by a Michaelis constant 30min before the reaction is carried out
This gives the GnT-II. DELTA. TM. mie constantIt was 55.98. mu.M.
Example 4: Fmoc-Asn-Gn2Man3 as substrate to verify the substrate specificity of GnT-II delta TM
The enzyme reaction system used for the assay was as follows (50. mu.L): 100mM MES/NaOH (pH 6.0), 10mM MnCl20.2. mu.M Fmoc-Asn-Gn2Man3, 0.2mM UDP-GlcNAc, 200. mu.g/mLGnT-II. DELTA. TM, the reaction was incubated at 37 ℃ for more than 5 h. And after the reaction is finished, centrifuging the reaction system at 15000r/min for 5min, taking the supernatant, detecting by high performance liquid chromatography, and comparing the reaction system with a standard sample.
As shown in FIG. 3B, only the substrate Fmoc-Asn-Gn2Man3 was detected in the reaction system, which was consistent with the elution time of the unreacted standard, indicating that GnT-II. DELTA.TM could not catalyze the addition of N-acetylglucosamine to Fmoc-Asn-Gn2Man 3.
Example 5: Fmoc-Asn-Gn2Man3GnGal is used as a substrate to verify the substrate specificity of GnT-II delta TM
The enzyme reaction system used for the assay was as follows (50. mu.L): 100mM MES/NaOH (pH 6.0), 10mM MnCl20.2 mu M Fmoc-Asn-Gn2Man3, 0.2mM UDP-GlcNAc, 0.2mM UDP-Galactose, 20. mu.g/mL GnT-I, 40. mu.g/mL GnalT, incubating the reaction system at 37 ℃ for more than 5h, inactivating the reaction system at 100 ℃ for 5min, adding 200. mu.g/mL GnT-II. delta. TM, and reacting at 37 ℃ for more than 5 h. And after the reaction is finished, centrifuging the reaction system at 15000r/min for 5min, taking the supernatant, detecting by high performance liquid chromatography, and comparing the reaction system with a standard sample.
As shown in FIG. 3C, Fmoc-Asn-Gn2Man3GnGal was detected in the reaction system, and no Fmoc-Asn-Gn2Man3Gn2Gal was detected which was further generated by the GnT-II. DELTA.TM-catalyzed reaction, indicating that GnT-II. DELTA.TM cannot catalyze the reaction of adding N-acetylglucosamine to Fmoc-Asn-Gn2Man3 GnGal.
Example 6: effect of reaction temperature on GnT-II. DELTA.TM protein Activity in vitro
The enzyme reaction system used for the assay was as follows (50. mu.L): 100mM MES/NaOH (pH 6.0), 10mM MnCl20.2. mu.M PA-Gn2Man3, 0.2mM UDP-GlcNAc, 20. mu.g/mL GnT-I. DELTA.TM, 200. mu.g/mL GnT-II. DELTA.TM, at 37 ℃ (16 ℃, 25 ℃, 30 ℃, 45 ℃, 50 ℃) for 30 min. The reaction is finishedAnd centrifuging the reaction system at 15000r/min for 5min, taking the supernatant, detecting by a high performance liquid chromatograph, and comparing the reaction system with a standard sample to obtain the reaction conversion rate at each temperature. The conversion of the reaction at 37 ℃ was defined as 1 unit, and the conversion at each temperature was compared therewith to obtain a relative conversion.
The results are shown in FIG. 5, where the GnT-II. DELTA. TM protein was 100% most reactive at 37 ℃.
Example 7: effect of reaction solution pH on GnT-II. DELTA. TM protein Activity in vitro
The enzyme reaction system used for the assay was as follows (50. mu.L): 100mM MES/NaOH (pH 3.5, 4.5, 5.5, 6.0, 6.5, 7.0, 7.5) or 100mM Tris/HCl (pH 7.5, 8.0, 8.5, 9.0), 10mM MnCl20.2. mu.M PA-Gn2Man3, 0.2mM UDP-GlcNAc, 20. mu.g/mL GnT-I. DELTA.TM, 200. mu.g/mL GnT-II. DELTA.TM, at 37 ℃ for 30 min. And after the reaction is finished, centrifuging the reaction system at 15000r/min for 5min, taking the supernatant, detecting by high performance liquid chromatography, and comparing the reaction system with a standard sample to obtain the reaction conversion rate in each pH solution. The reaction conversion at pH6.0 was defined as 1 unit, and the conversion at each pH was compared with it to obtain a relative conversion.
As shown in FIG. 6, GnT-II. DELTA.TM gave the highest reaction conversion at pH 7.0, and showed higher activity at pH 6.0-7.5.
Example 8: effect of divalent cation type on the in vitro Activity of GnT-II. DELTA.TM proteins
The enzyme reaction system used for the assay was as follows (50. mu.L): 100mM MES/NaOH (pH 6.0), 10mM MnCl2(CoCl2、MgCl2、NiCl2、CaCl2、CuCl2、FeCl2) Or EDTA, 0.2. mu.M PA-Gn2Man3, 0.2mM UDP-GlcNAc, 20. mu.g/mL GnT-I. DELTA. TM, 200. mu.g/mL GnT-II. DELTA. TM, at 37 ℃ for 30 min. And after the reaction is finished, centrifuging the reaction system at 15000r/min for 5min, taking supernate, detecting by high performance liquid chromatography, and comparing the reaction system with a standard sample to obtain the reaction conversion rate in each divalent cation solution.
The reaction solution contains Mn2+The conversion of the reaction at time is defined as 1 unit, and the conversion of each divalent cation solution is compared therewith to obtain a relative conversion. The results are shown in FIG. 7, where Mn was added to the reaction solution of GnT-II. DELTA.TM protein2+The activity is the greatest in the case of adding Co2+Or Mg2+It is also active.
Example 9: effect of divalent manganese ion concentration on GnT-II. DELTA.TM protein Activity in vitro
The enzyme reaction system used for the assay was as follows (50. mu.L): 100mM MES/NaOH (pH 6.0), 10mM (0.1mM, 0.5mM, 1mM, 2mM, 5mM, 20mM) MnCl20.2. mu.M PA-Gn2Man3, 0.2mM UDP-GlcNAc, 20. mu.g/mL GnT-I. DELTA.TM, 200. mu.g/mL GnT-II. DELTA.TM, at 37 ℃ for 30 min. And after the reaction is finished, centrifuging the reaction system at 15000r/min for 5min, taking the supernatant, detecting by high performance liquid chromatography, and comparing the reaction system with a standard sample to obtain the reaction conversion rates in the solutions with different manganese ion concentrations.
The reaction solution contained 10mM Mn2+The reaction conversion is defined as 1 unit, different Mn2+The conversion in the concentration solution was compared to obtain the relative conversion. The results are shown in FIG. 8, where the GnT-II. DELTA. TM protein was added to the reaction at 10mM Mn2+The activity is the greatest in the case of (2), on the basis of which more Mn is added2+Does not increase the activity of the protein.
Example 10:
the pesticide-GnT-II is expressed by adding a pesticide tag, FIG. 9 shows the experimental result of the pesticide-GnT-II by adding a pesticide tag, and as shown in FIG. 9, the pesticide-GnT-II expresses the inclusion body protein, so that prokaryotic expression cannot be successfully realized.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A prokaryotic expression method of human N-acetylglucosamine transferase II is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
prokaryotic expression of GnT-II: expressing human GnT-II delta TM in escherichia coli, wherein the GnT-II delta TM is human N-acetylglucosamine transferase II with 1-87 amino acids at the N end cut off;
purification of GnT-II: purifying the prokaryotically expressed GnT-II delta TM;
1-87 amino acids at the N end of GnT-II are cut off to construct a prokaryotic expression vector;
transforming prokaryotic host bacteria, coating on a plate culture medium, and standing overnight;
screening single colonies of host bacteria, inoculating the single colonies to an LB liquid culture medium, and carrying out shaking culture overnight to obtain a bacterial liquid;
inoculating the bacterial liquid to a TB liquid culture medium, carrying out shake culture, and then carrying out cooling culture;
adding an inducer, carrying out shake culture, centrifugally collecting thalli, suspending in a buffer solution, carrying out ultrasonic crushing, centrifuging, and collecting a supernatant;
and (3) washing the affinity chromatography column by using the buffer solution, loading the supernatant, and collecting the eluent to obtain the human N-acetylglucosamine transferase II.
2. The method for prokaryotic expression of human N-acetylglucosaminyltransferase II according to claim 1, wherein: the GnT-II is GnT-II delta TM, aa 88-443; the prokaryotic expression vector is pET28a-GnT-II delta TM; the transformed prokaryotic host bacterium is ROSETTA.
3. The method for prokaryotic expression of the human N-acetylglucosaminyltransferase II according to any one of claims 1 or 2, wherein: the plate culture medium is LB, kanamycin and chloramphenicol plate culture medium; the LB liquid culture medium is LB, kanamycin and chloramphenicol liquid culture medium; the TB liquid culture medium is TB, kanamycin and chloramphenicol; the buffer solution is Tris/HCl and NaCl buffer solution; the inducer is IPTG; the affinity chromatographic column is a nickel ion affinity chromatographic column.
4. The method for prokaryotic expression of human N-acetylglucosaminyltransferase II according to claim 3, wherein: the buffer solution has the concentration of 25mM of Tris/HCl and 150mM of NaCl respectively.
5. The method for prokaryotic expression of human N-acetylglucosaminyltransferase II according to claim 1, 2 or 4, wherein: the inducer concentration is 0.1 mM.
6. The method for prokaryotic expression of human N-acetylglucosaminyltransferase II according to claim 5, wherein: the pH of the buffer was 8.0.
7. The method for prokaryotic expression of human N-acetylglucosaminyltransferase II according to claim 1, 2 or 6, wherein: the strain is inoculated in an LB liquid culture medium, and is subjected to shaking culture overnight, wherein the culture temperature is 37 ℃; inoculating the bacterial liquid to a TB liquid culture medium, carrying out shake culture, and then carrying out cooling culture, wherein the temperature is 37 ℃, the oscillation rate is 200r/min, the oscillation time is 3h, the temperature is reduced to 16 ℃, and the cooling culture time is 1 h; adding an inducer, and carrying out shaking culture, wherein the frequency of the shaking culture is 200r/min, and the culture time is 20 h; the thalli is collected by centrifugation, the centrifugation speed is 9000r/min, and the centrifugation time is 60 min.
8. The method for prokaryotic expression of human N-acetylglucosaminyltransferase II according to claim 7, wherein: inoculating the bacterial liquid to a TB liquid culture medium, carrying out shake culture, and then carrying out cooling culture to obtain bacterial liquid OD6000.6 to 0.8.
9. The method for prokaryotic expression of human N-acetylglucosaminyltransferase II according to claim 1, 2 or 8, wherein: and washing the affinity chromatography column by using the buffer solution, loading the supernatant, and collecting eluent, wherein the volume ratio of the buffer solution to the affinity chromatography column is 10: 1, and the flow rate of the buffer solution is 1 min/mL.
10. The human N-acetylglucosamine transferase II produced by the method according to claim 1 to 9, wherein: the michaelis constant of the human N-acetylglucosamine transferase II is 55.98 mu M.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112375810A (en) * | 2020-11-16 | 2021-02-19 | 北京大学人民医院 | Application of GnT-II gene down-regulated expression as liver cancer prognosis marker |
| WO2021227930A1 (en) * | 2020-05-14 | 2021-11-18 | 江南大学 | Method for improving soluble expression of recombinant n-acetylglucosaminyl transferase ii |
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| JP2001178453A (en) * | 1999-12-24 | 2001-07-03 | Toyobo Co Ltd | Method for producing n-acetylglucosaminyltransferase |
| CN103589695A (en) * | 2013-09-30 | 2014-02-19 | 安徽丰原发酵技术工程研究有限公司 | N-acetyl glucosamine transferase mutant and application thereof |
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| JP2001178453A (en) * | 1999-12-24 | 2001-07-03 | Toyobo Co Ltd | Method for producing n-acetylglucosaminyltransferase |
| CN103589695A (en) * | 2013-09-30 | 2014-02-19 | 安徽丰原发酵技术工程研究有限公司 | N-acetyl glucosamine transferase mutant and application thereof |
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| KAZUHITO FUJIYAMA ET AL.: "Human N-acetylglucosaminyltransferase I. Expression in Escherichia coli as a soluble enzyme, and application as an immobilized enzyme for the chemoenzymatic synthesis of N-linked oligosaccharides", 《J BIOSCI BIOENG》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021227930A1 (en) * | 2020-05-14 | 2021-11-18 | 江南大学 | Method for improving soluble expression of recombinant n-acetylglucosaminyl transferase ii |
| CN112375810A (en) * | 2020-11-16 | 2021-02-19 | 北京大学人民医院 | Application of GnT-II gene down-regulated expression as liver cancer prognosis marker |
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