CN108179160B - Preparation method of high mannose type oligosaccharide connected by phytol - Google Patents
Preparation method of high mannose type oligosaccharide connected by phytol Download PDFInfo
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Abstract
The invention discloses a preparation method of high mannose type oligosaccharide connected by phytol, which comprises the following steps of prokaryotic expression of mannosyltransferase: respectively expressing yeast-derived Alg1 Delta TM, Trx-Alg2, Alg11 Delta TM, Dpm1, M-Alg3, M-Alg9 and Alg12 in Escherichia coli; purifying the mannosyltransferase; preparing a mannosyltransferase membrane component; synthesizing a donor substrate PP-Man; PPGn2‑Man1、PPGn2‑Man3、PPGn2‑Man5、PPGn2‑Man6、PPGn2‑Man7、PPGn2‑Man8、PPGn2‑Man9And PPGn2‑Man4And (4) synthesizing.The invention purifies yeast-derived Alg1 delta TM, Trx-Alg2, Alg11 delta TM, Dpm1, M-Alg3, M-Alg9 and Alg12 mannosyltransferase by prokaryotic expression, and successfully prepares PPGn in vitro2‑Man1、PPGn2‑Man3、PPGn2‑Man5、PPGn2‑Man6、PPGn2‑Man7、PPGn2‑Man8、PPGn2‑Man9And PPGn2‑Man4The high mannose type oligosaccharide solves the technical problems that multiple transmembrane eukaryotic protein prokaryotic expression is easy to degrade and eukaryotic membrane protein expression and purification are difficult, and the high efficiency and stereoselectivity of mannosyltransferase overcome the difficulty of organic synthesis and promote the development of glycochemistry and glycobiology.
Description
Technical Field
The invention belongs to the technical field of molecular biology and biochemistry, and particularly relates to a preparation method of high mannose type oligosaccharide connected by phytol.
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. High mannose type oligosaccharides are widely used in biological research, such as sugar chips, sugar vaccines, and protein amount control, as a large group of N-glycosylation modifications. At present, the high mannose type oligosaccharide is mainly prepared by a chemical method and is extracted from plants or yeast, however, the two methods are time-consuming and labor-consuming, the byproducts are many and the yield is extremely low. The chemoenzymatic method, as an alternative to chemical synthesis, has the advantages of high stereoisomeric selectivity and extremely high conversion rate, and is currently widely used for polysaccharide synthesis. However, no chemical enzyme method for synthesizing high mannose type oligosaccharides has been reported at present, and the main reason is that a series of enzymes for catalytically synthesizing the structure are endoplasmic reticulum resident enzymes which are all eukaryotic membrane proteins, and the expression and purification are extremely difficult; another reason is that the substrate for the enzyme is a lipid phosphate linked sugar, which is not readily available.
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, the invention overcomes the defects in the prior art and provides a preparation method of the high mannose type oligosaccharide connected by the phytol.
In order to solve the technical problems, the invention provides the following technical scheme: a method for the preparation of a phytanol-linked high mannose oligosaccharide, comprising prokaryotic expression of a mannosyltransferase: respectively expressing yeast-derived Alg1 Δ TM, Trx-Alg2, Alg11 Δ TM, Dpm1, M-Alg3, M-Alg9 and Alg12 in Escherichia coli, wherein the Alg1 Δ TM is a mannosyltransferase Alg1 with an N-terminal transmembrane domain truncated, the Trx-Alg2 is a mannosyltransferase Alg2 with a Trx tag fused, the Alg11 TM is a mannosyltransferase Alg11 with an N-terminal transmembrane domain truncated, the Dpm1 is a long-base phospho-mannosyltransferase polypeptide 1, the M-Alg3 is a mannosyltransferase Alg3 with a Mistic tag fused, the M-Alg9 is a mannosyltransferase Alg9 with a Mistic tag fused, and the Alg12 is a mannosyltransferase Alg 12; purification of mannosyltransferase: respectively purifying the Alg1 Δ TM, Trx-Alg2, Alg11 Δ TM and Dpm1 which are subjected to prokaryotic expression; preparation of mannosyltransferase Membrane component: respectively preparing membrane components from the M-Alg3, M-Alg9 and Alg12 which are subjected to prokaryotic expression; synthesis of the donor substrate PP-Man: synthesizing the PP-Man by catalyzing phytonyl-phosphate and donor GDP-Man by the purified Dpm1 protein; PPGn2-Man1、PPGn2-Man3And PPGn2-Man5The synthesis of (2): sequentially obtaining the PPGn by catalyzing the PPGn2 and the GDP-Man by the mannosyltransferases Alg1 Δ TM, Trx-Alg2 and Alg11 Δ TM2-Man1、PPGn2-Man3And PPGn2-Man5；PPGn2-Man6、PPGn2-Man7、PPGn2-Man8And PPGn2-Man9The synthesis of (2): synthesizing the PPGn2-Man5Adding PP-Man into the reaction system, and sequentially obtaining PPGn under the catalysis of the mannosyltransferases M-Alg3, M-Alg9 and M-Alg122-Man6、PPGn2-Man7、PPGn2-Man8And PPGn2-Man9(ii) a PPGn of unusual structure2-Man4、PPGn2-Man5、PPGn2-Man6And PPGn2-Man7The synthesis of (2): synthesizing the PPGn2-Man3Adding PP-Man into the reaction system, and sequentially obtaining PPGn with an unusual structure under the catalysis of the mannosyltransferases M-Alg3, M-Alg9 and M-Alg122-Man4、PPGn2-Man5、PPGn2-Man6And PPGn2-Man7。
As a preferable scheme of the preparation method of the phytanol-linked high mannose type oligosaccharide, the method comprises the step of prokaryotic expression of the mannosyltransferase, wherein the escherichia coli is a ROSETTA prokaryotic expression host bacterium.
As a preferred scheme of the preparation method of the phytanol-linked high mannose type oligosaccharide, the prokaryotic expression is carried out by the following vectors: pET28a-Alg1DTM, pET32a-Trx-Alg2, pET28a-Alg11DTM, pET28a-Dpm1, pET28a-M-Alg3, pET28a-M-Alg9 and pET28a-Alg 12.
The purification of the mannosyltransferase is a preferred scheme of the preparation method of the phytanol-linked high mannose oligosaccharide, wherein recombinant escherichia coli with prokaryotic expression of Alg1DTM, Trx-Alg2 and Dpm1 is respectively subjected to ultrasonic disruption, Alg1DTM, Trx-Alg2 and Dpm1 membrane components are respectively prepared by centrifugation, and the Alg1DTM, Trx-Alg2 and Dpm1 membrane components are respectively dissolved in 1% Triton X-100 buffer solution to obtain Alg1DTM, Trx-Alg2 and Dpm1 membrane proteins; carrying out ultrasonic crushing on recombinant escherichia coli of which the prokaryotic expression is Alg11DTM, and then centrifuging to obtain Alg11DTM supernatant protein; respectively purifying the Alg1DTM, Trx-Alg2 and Dpm1 membrane proteins by nickel ion affinity chromatography, and purifying the Alg11DTM supernatant protein by nickel ion affinity chromatography.
As a preferred scheme of the preparation method of the phytanol-linked high mannose oligosaccharide, Alg1DTM, Trx-Alg2 and Dpm1 membrane components are prepared by centrifugation respectively, wherein recombinant escherichia coli of which prokaryotic expression is Alg1DTM, Trx-Alg2 and Dpm1 is subjected to ultrasonic disruption respectively, then is centrifuged at 4000r/min for 20 min, sediment is discarded, supernatant is collected, and is centrifuged at 15000r/min for 90 min at high speed, and the supernatant is discarded; and centrifuging to obtain Alg11DTM supernatant protein, wherein the centrifugation speed is 15000r/min, and the centrifugation time is 90 min.
As a preferred scheme of the preparation method of the phytanol-linked high mannose type oligosaccharide, the preparation method of the mannosyltransferase membrane component comprises the steps of respectively carrying out ultrasonic crushing on recombinant escherichia coli of which prokaryotic expression is expressed by M-Alg3, M-Alg9 and Alg12, centrifuging at 4000r/min for 20 min, discarding a precipitate, collecting a supernatant, carrying out ultracentrifugation, discarding the supernatant to obtain an escherichia coli cell membrane component, wherein the ultracentrifugation speed is 100000r/min, and the time is 60 min.
As a preferable scheme of the preparation method of the phytanol-linked high mannose oligosaccharide, the donor substrate PP-Man is synthesized, wherein purified Dpm1 protein catalyzes phytanyl-phosphate and donor GDP-Man, and the reaction system of the PP-Man comprises the following components: 1 mM/muL Tris/HCl pH7.5, 0.2 mM/muL MgCl 21% NP-40, 0.4 mM/. mu.L phytonyl-phosphate, 1 mM/. mu.L GDP-Man and 2 mg/mL Dpm1, wherein the reaction temperature is 30 ℃ and the reaction time is 10 hours.
As a preferable embodiment of the method for producing a phytol-linked high mannose type oligosaccharide according to the present invention, the PPGn is a peptide of the present invention2-Man1、PPGn2-Man3And PPGn2-Man5The reaction system is as follows: 14 mM/60 muL MES pH 6.0, 4mM/60 muL Lpotassium citrate, 10mM/60 muL MgCl2, 10mM/60 muL MnCl2, 0.05% NP-40, 50M/60 muL PPGn2, 1M/60 muL sucrose, 10 muL PE liposome and 2 mM/60 muL GDP-Man are sequentially added into the reaction system, 0.5g/mLAlg1 Tam TM, 150 mug/mL Trx-Alg2 and 50 mug/mLAlg 11 TM are sequentially added into the reaction system, the reaction temperature is 30 ℃, the reaction time is 12 hours,sequentially obtaining PPGn2-Man1、PPGn2-Man3、PPGn2-Man5。
As a preferable embodiment of the method for producing a phytol-linked high mannose-type oligosaccharide of the present invention, the method is directed to the synthesis of PPGn2-Man5Adding 2mM PP-Man into the reaction system, and sequentially adding 20mg/mL M-Alg3, 20mg/mL M-Alg9 and 20mg/mL Alg12 to sequentially obtain PPGn2-Man6、PPGn2-Man7、PPGn2-Man8And PPGn2-Man9。
As a preferable embodiment of the method for producing a phytol-linked high mannose-type oligosaccharide of the present invention, the method is directed to the synthesis of PPGn2-Man3Adding 2mM PP-Man into the reaction system, sequentially adding 20mg/mL M-Alg3, 20 mg/mLM-Alg9 and 20mg/mL Alg12, reacting at 30 ℃ for 12 hours, and sequentially obtaining the PPGn with the unusual structure2-Man4、PPGn2-Man5、PPGn2-Man6And PPGn2-Man7。
The invention has the beneficial effects that: the invention prokaryotic expresses Alg1 Δ, Trx-Alg2, Alg11 Δ, Dpm1, M-Alg3, M-Alg9 and Alg12 mannosyltransferase which are from purified yeast, and the PPGn is successfully prepared in vitro2-Man1、PPGn2-Man3、PPGn2-Man5、PPGn2-Man6、PPGn2-Man7、PPGn2-Man8、PPGn2-Man9And PPGn2-Man4The high mannose type oligosaccharide solves the technical problems that multiple transmembrane eukaryotic protein prokaryotic expression is easy to degrade and eukaryotic membrane protein expression and purification are difficult, and the high efficiency and stereoselectivity of mannosyltransferase overcome the difficulty of organic synthesis and promote 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 Alg1, Alg2, and Alg11 (FIG. 1A); prediction of transmembrane topology of Alg3, Alg9, and Alg12 (fig. 1B).
FIG. 2 is a SDS-PAGE Coomassie brilliant blue image of protein electrophoresis of Saccharomyces cerevisiae Alg1DTM, Trx-Alg2, Alg11DTM and Dpm1 purified proteins (FIG. 2A); western blotting (western blotting) images of prokaryotic expression of Alg3 and Alg9, Vector representing control (fig. 2B); FIG. 2C is a western blotting (Western blotting) image of M-Alg3, M-Alg9, and Alg12 prokaryotic expression, with Vector representing the control (FIG. 2C).
FIG. 3 shows the chemical enzymatic synthesis of PPGn2-Man1、PPGn2-Man3、PPGn2-Man5A flow chart (FIG. 3A); substrate PPGn2Respectively adding Alg1DTM, Trx-Alg2 and Alg11DTM in sequence to synthesize PPGn in sequence2-Man1、PPGn2-Man3And PPGn2-Man5Three structural diagrams (fig. 3B).
FIG. 4 shows the chemical enzymatic synthesis of PPGn2-Man5To PPGn2-Man9A flow chart (fig. a); substrate PPGn2-Man5Respectively adding M-Alg3, M-Alg3, M-Alg9, M-Alg3 and M-Alg9, reacting for 12 hours, adding Alg12, simultaneously adding M-Alg3, M-Alg9 and Alg12, and sequentially synthesizing PPGn2-Man6、PPGn2-Man7、PPGn2-Man8And PPGn2-Man9(FIG. B).
FIG. 5 shows the final product PPGn2-Man9ESI-MS after acid hydrolysis purification shows the results of determination of the molecular weight (FIG. 5A); PPGn2-Man9Generating PPGn after alpha 1,2 Mannosidase treatment2-Man5(ii) a The PPGn is generated after alpha 1-2,3 Mannosidase treatment2-Man3Simultaneously processing alpha 1-2,3 Mannosidase and alpha 1,6 Mannosidase to generate PPGn2-Man1The PPGn is generated after b-Mannosidase treatment2(FIG. 5B).
FIG. 6 is a chemical enzymatic synthesis of PPGn2-Man4、PPGn2-Man5、PPGn2-Man6And PPGn2-Man7Scheme for four unusual high mannose oligosaccharides (fig. 6A). B is to PPGn2-Man3Adding 2mM PP-Man into the reaction system, sequentially adding 20mg/mL M-Alg3, 20 mg/mLM-Alg9 and 20mg/mL Alg12, reacting at 30 ℃ for 12 hours, and sequentially obtaining the PPGn with the unusual structure2-Man4、PPGn2-Man5、PPGn2-Man6And PPGn2-Man7(FIG. 6B).
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.
Reagents referred to in the examples: restriction enzymes, Taq DNA polymerase, ligase and the like were purchased from Nippon Biotechnology Ltd (Dalian), gel recovery, PCR product purification and plasmid extraction kits and IPTG were purchased from Shanghai, PC was purchased from Chinese medicine, GDP-Man and α 1-2-Mannosidase were purchased from Sigma-Aldrich, α 1-2, 3-and α 1, 6-Mannosidase were purchased from New England Biolabs, α 1, 2-Mannosidase were purchased from ProZyme. Primer synthesis and sequencing are both in Huada gene.
Example 1: prokaryotic expression purification of Saccharomyces cerevisiae Alg1DTM, Trx-Alg2, Alg11DTM and Dpm1
We analyzed the membrane topology of the three eukaryotic transmembrane proteins yeast Alg1, Alg2, Alg11 with the software (TMHMM Server v.2.0) (FIG. 1A). The N-end transmembrane structure (Alg 1DTM, aa 35-349; Alg11DTM, aa 45-548) is cut off by Alg1 and Alg 11; alg2 fused with Trx tag (Trx-Alg 2) to construct the following prokaryotic expression vector: pET28a-Alg1DTM, pET32a-Trx-Alg2 and pET28a-Alg11DTM, and vector pET28a-Dpm1 was constructed. The recombinant prokaryotic expression plasmid is transformed into a ROSETTA prokaryotic expression host bacterium, a single colony is picked from a transformation plate on the day next to LB + (Kan or Amp) + chloramphenicol (34 mu g/mL), and is inoculated into 5mL of LB + (Kan or Amp) + chloramphenicol liquid culture medium and is cultured with shaking at 37 ℃ overnight. Inoculating 2mL of overnight cultured bacterial liquid into 200mL of TB + (Kan or Amp) + chloramphenicol liquid culture medium, culturing at 37 deg.C under shaking at 200r for 3 hr to make OD600 reach 0.6-0.8, transferring to 16 deg.C, culturing for 1 hr, adding IPTG to make final concentration reach 0.1 mmol/L, and performing induction culture at 200 r/min for 20 hr. The cells were collected by centrifugation, resuspended in 20 mL of A buffer (25 mM Tris/HCl (pH 8.0), 150 mM NaCl), sonicated to fragment pET28a-Alg1DTM, pET32a-Trx-Alg2 and pET28a-Dpm1 recombinant bacteria, centrifuged at 4000r/min for 20 min, the pellet was discarded, the supernatant was collected by high speed centrifugation at 15000r/min for 90 min, the supernatant was discarded, the pellet was resuspended in 10 mL of B buffer (25 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100) at 4 ℃ for 30 min and then centrifuged at 12000r/min for 30 min, and the supernatant was collected for purification. In addition, the pET28a-Alg11DTM recombinant bacteria are subjected to ultrasonic disruption and then are centrifuged at a high speed of 15000r/min for 90 min, and supernatant protein is taken for purification.
Alg1DTM, Trx-Alg2 and Dpm1 protein purification: the HisTrap HP 1mL affinity chromatography column was equilibrated with a 10-fold volume flow rate of solution B for 1min/mL, and then about 10 mL of the supernatant was applied, followed by washing the column with 10 mL of solutions B containing 20, 60, and 500mM imidazole, respectively, and collecting the washed solutions in a centrifuge tube. Each sample was examined by SDS-PAGE electrophoresis. Alg11DTM purification was consistent with the above except that each buffer contained no Triton X-100. FIG. 2A shows the successful purification of the above-described mannosyltransferase.
Example 2: saccharomyces cerevisiae M-Alg3, M-Alg9 and Alg12 prokaryotic expression and membrane component preparation
The membrane topologies of the three eukaryotic transmembrane proteins yeast Alg3, Alg9, Alg12 were analyzed with software (TMHMM Server v.2.0) (fig. 1B), constructing a prokaryotic vector: pET28a-Alg3, pET28a-Alg9 and pET28a-Alg12, protein immunoblotting (western blotting) detection shows that both Alg3 and Alg9 are degraded (figure 2B), Mistic is fused at the N ends of Alg3 and Alg9, and a prokaryotic expression vector is constructed: pET28a-M-Alg3 and pET28a-M-Alg9, firstly, corresponding genes are amplified from the genome of Saccharomyces cerevisiae W303A and are respectively connected into a prokaryotic expression vector to construct a recombinant vector: pET28a-M-Alg3, pET28a-M-Alg9 and pET28a-Alg 12. The recombinant prokaryotic expression plasmid is transformed into a ROSETTA prokaryotic expression host bacterium, and the subsequent protein induction expression is consistent with the protein of the example 1. Collecting 10 mL of recombinant Escherichia coli cultured for 20 hr under induction, resuspending in 0.5 mL buffer (25 mM Tris/HCl (pH 8.0), 150 mM NaCl), ultrasonicating, centrifuging at 4000r/min for 20 min, discarding the precipitate, collecting supernatant, ultracentrifuging at 100000r/min for 60 min, discarding the supernatant, and resuspending the precipitate in 100L buffer (50 mM MES, pH 6.5, 30% glycerol) to obtain the cell membrane fraction of Escherichia coli. The results of western blotting (western blotting) are shown in FIG. 2C, and all three recombinant proteins were expressed efficiently.
Example 3: enzymatic Synthesis of phytonyl-phosphate-Man (PP-Man)
The standard enzyme reaction conditions were as follows (50 μ L system): 50 mM Tris/HCl (pH 7.5), 10mM MgCl21% NP-40, 20 mM phytonyl-phosphate, 50 mM GDP-Man and 2 mg/mL purified Dpm1 protein. The reaction was incubated at 30 ℃ for 10 hours. The efficiency of the enzyme reaction was monitored by TLC.
Example 4: synthesis of phytol-linked high mannose oligosaccharide PPGn by chemical enzyme method2-Man1、PPGn2-Man3、PPGn2-Man5
The standard enzyme reaction conditions were as follows (60 μ L system): 14 mM MES (pH 6.0), 4mM potassium citrate, 10 mM MgCl2, 10 mM MnCl20.05% NP-40, 50 mM PPGn2, 1M sucrose, 10 μ L of PE liposomes and 2mM GDP-Man. As shown in the flow chart of FIG. 3A, for the enzymatic synthesis of PPGn2-Man1-5Several structures, the amount of purified mannosyltransferase added to the above reaction system is: alg1DTM (0.5mg/mL), Trx-Alg2 (150 μ g/mL) and Alg11DTM (50 μ g/mL), the reaction was incubated at 30 ℃ for 12 hours. After the reaction, 0.2mL of 20 mM HCl was added thereto and the mixture was subjected to acidolysis at 100 ℃ for 1 hour to release the sugar chains and the fatty chains Phytanol. The acidolysis product was centrifuged at 12000r/min for 1min, the supernatant was purified by 1mL of solid phase extraction packing Supelclean ENVI-Carb Slurry, and the sugar chain component was eluted with 3mL of 25% acetonitrile and then lyophilized. The purified sugar chain powder finally obtained was dissolved in 40 mL of pure water, and 1mL of the purified sugar chain powder was subjected to autoinjection by a Dionex Ultimate 3000 UPLC (Thermoscientific) chromatograph in an amino chromatographic Column (Waters Acquity UPLC BEH Amide Column 1.7 mm 2.1X 100 mm), and subjected to acetonitrile linear gradient elution (solution A: CH3 CN; solution B: H2O; elution conditions: 0-2 min, 20% B; 2-15 min, 20-50% B; 15-18 min, 50% B; flow rate: 0.2 mL/min) to separate the substrate from the product. The effluent was simultaneously tested for molecular weight by ESI-MS instrument TSQ Quantum Ultra (ThermoScientific) (scan 400-.
Results as shown in fig. 3B, we were able to synthesize PPGn sequentially by sequential addition of mannosyltransferases2-Man1、PPGn2-Man3And PPGn2-Man5The molecular weights of all three structures, determined by ESI-MS, were in full agreement with the predictions.
Example 5: synthesis of phytol-linked high mannose oligosaccharide PPGn by chemical enzyme method2-Man6-9
As shown in the flow chart of FIG. 4A, for the enzymatic synthesis of PPGn2-Man6-9Several structures to synthesize the PPGn2-Man5Adding 2mM PP-Man into the reaction system, adding M-Alg3, or simultaneously adding M-Alg3 and M-Alg9, or adding M-Alg3 and M-Alg9 firstly, reacting for 12 hours, then adding Alg12, or simultaneously addingAdding M-Alg3, M-Alg9 and Alg12, and respectively synthesizing PPGn in sequence2-Man6、PPGn2-Man7、PPGn2-Man8Or PPGn2-Man9Sequentially obtaining PPGn2-Man6、PPGn2-Man7、PPGn2-Man8And PPGn2-Man9The concentration of the M-Alg3 is 20mg/mL, the concentration of the M-Alg9 is 20mg/mL, the concentration of the Alg12 is 20mg/mL, and the reaction is incubated at 30 ℃. The workup after the end of the reaction and its LC-MS conditions were in accordance with example 4. Results as shown in fig. 4B, we were able to synthesize PPGn sequentially by sequential addition of mannosyltransferases2-Man6、PPGn2-Man7、PPGn2-Man8And PPGn2-Man9Four high mannose type oligosaccharide structures, all of which were determined for their molecular weight by ESI-MS, were in full agreement with the predictions.
Example 6: high mannose type oligosaccharide end product (Gn)2-Man9) Identification of the three-dimensional Structure of
The ESI-MS results of FIG. 5A show the final product Gn2-Man9The molecular weight after sodium addition was 1907.86, consistent with the prediction. In order to further determine Gn2-Man9The final product is added with different mannose hydrolase respectively. The reaction system is as follows: for α 1-2,3 manosinase and α 1,6 manosinase: 1mL of commercial buffer (10X), 1mL of BSA (10X), 8 mL of oligosaccharide (2.5 nmol), 0.1 mL of enzyme (3.2 and 4U, respectively) reacted at 25 ℃ for 16 hr; for α 1,2 manosidase: 2mL of commercial buffer (5X), 8 mL of oligosaccharide (2.5 nmol), 0.1 mL of enzyme (0.1 mU), and reaction at 25 ℃ for 16 hr; for b-Mannosidase: 50 mM sodium citrate buffer (pH 4.4), 8 mL oligosaccharide (2.5 nmol), 0.1U enzyme, 25 ℃ reaction for 16 hr. Results Gn is shown in the results of FIG. 5B2-Man9Gn is generated after alpha 1,2 Mannosidase treatment2-Man5(ii) a Gn is generated after alpha 1-2,3 Mannosidase treatment2-Man3(ii) a Simultaneously, after alpha 1-2,3 Mannoside and alpha 1,6 Mannoside treatment, Gn is generated2-Man1(ii) a Gn is generated after b-Mannosidase treatment2. In conclusion, the spatial structure of the product synthesized by the in vitro chemical enzyme method is consistent with the structure in the cell.
Example 7: chemical enzyme method for synthesizing unusual high mannose type oligosaccharide
As shown in the flow chart of fig. 6A, with PPGn2-Man3Adding 2mM PP-Man and colibacillus membrane components containing recombinant mannosyltransferase into a reaction system as substrates, wherein the components are respectively as follows: M-Alg3 (20 mg/mL), M-Alg9 (20 mg/mL) and Alg12 (20 mg/mL), the reaction was incubated at 30 ℃ for 12 hours. Results as shown in fig. 6B, we were able to synthesize PPGn sequentially by sequential addition of mannosyltransferases2-Man4、PPGn2-Man5、PPGn2-Man6And PPGn2-Man7Four high mannose oligosaccharides of unusual structure as shown.
The invention prokaryotic expresses Alg1 Δ, Trx-Alg2, Alg11 Δ, Dpm1, M-Alg3, M-Alg9 and Alg12 mannosyltransferase which are from purified yeast, and the PPGn is successfully prepared in vitro2-Man1、PPGn2-Man3、PPGn2-Man5、PPGn2-Man6、PPGn2-Man7、PPGn2-Man8、PPGn2-Man9And PPGn2-Man4The high mannose type oligosaccharide solves the technical problems that multiple transmembrane eukaryotic protein prokaryotic expression is easy to degrade and eukaryotic membrane protein expression and purification are difficult, and the high efficiency and stereoselectivity of mannosyltransferase overcome the difficulty of organic synthesis and promote the development of glycochemistry and glycobiology.
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 (7)
1. A method for preparing high mannose type oligosaccharide connected by phytanol is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
prokaryotic expression of mannosyltransferase: respectively expressing yeast-derived Alg1 Δ TM, Trx-Alg2, Alg11 Δ TM, Dpm1, M-Alg3, M-Alg9 and Alg12 in Escherichia coli, wherein the Alg1 Δ TM is a mannosyltransferase Alg1 with an N-terminal transmembrane domain truncated, the Trx-Alg2 is a mannosyltransferase Alg2 with a Trx tag fused, the Alg11 TM is a mannosyltransferase Alg11 with an N-terminal transmembrane domain truncated, the Dpm1 is a long-base phospho-mannosyltransferase polypeptide 1, the M-Alg3 is a mannosyltransferase Alg3 with a Mistic tag fused, the M-Alg9 is a mannosyltransferase Alg9 with a Mistic tag fused, and the Alg12 is a mannosyltransferase Alg 12;
purification of mannosyltransferase: respectively purifying the Alg1 Δ TM, Trx-Alg2, Alg11 Δ TM and Dpm1 which are subjected to prokaryotic expression;
preparation of mannosyltransferase Membrane component: respectively preparing membrane components from the M-Alg3, M-Alg9 and Alg12 which are subjected to prokaryotic expression;
synthesis of the donor substrate PP-Man: synthesizing the PP-Man by catalyzing phytonyl-phosphate and donor GDP-Man by the purified Dpm1 protein;
PPGn2-Man1、PPGn2-Man3and PPGn2-Man5The synthesis of (2): the reaction system is 14 mM/60 muL MES pH 6.0, 4mM/60 muL Lpotassiumcrate, 10mM/60 muL MgCl2, 10mM/60 muL MnCl2, 0.05% NP-40, 50M/60 muL PPGn21M/60 mu L sucrose, 10 mu L/60 mu L PE liposome and 2 mM/60 mu L GDP-Man are sequentially added into the reaction system, 0.5g/mLAlg1 TM, 150 mu g/mL Trx-Alg2 and 50 mu g/mLAlg11 TM are sequentially added into the reaction system, the reaction temperature is 30 ℃, the time is 12 hours, and PPGn is sequentially obtained2-Man1、PPGn2-Man3、PPGn2-Man5；
PPGn2-Man6、PPGn2-Man7、PPGn2-Man8And PPGn2-Man9The synthesis of (2): to synthesize PPGn2-Man5Reaction of (2)Adding 2mM PP-Man into the system, adding M-Alg3, or simultaneously adding M-Alg3 and M-Alg9, or adding M-Alg3 and M-Alg9 firstly, reacting for 12 hours, then adding Alg12, or simultaneously adding M-Alg3, M-Alg9 and Alg12, and sequentially and respectively synthesizing PPGn2-Man6、PPGn2-Man7、PPGn2-Man8Or PPGn2-Man9Sequentially obtaining PPGn2-Man6、PPGn2-Man7、PPGn2-Man8And PPGn2-Man9The concentration of the M-Alg3 is 20mg/mL, the concentration of the M-Alg9 is 20mg/mL, and the concentration of the Alg12 is 20 mg/mL;
PPGn of unusual structure2-Man4、PPGn2-Man5、PPGn2-Man6And PPGn2-Man7The synthesis of (2): to synthesize PPGn2-Man3Adding 2mM PP-Man into the reaction system, sequentially adding 20mg/mL M-Alg3, 20 mg/mLM-Alg9 and 20mg/mL Alg12, reacting at 30 ℃ for 12 hours, and sequentially obtaining the PPGn with the unusual structure2-Man4、PPGn2-Man5、PPGn2-Man6And PPGn2-Man7。
2. The method of claim 1, wherein: prokaryotic expression of the mannosyltransferase is carried out, wherein the escherichia coli is a ROSETTA prokaryotic expression host bacterium.
3. The method of claim 1 or 2, wherein: the vectors of the prokaryotic expression are respectively as follows: pET28a-Alg1DTM, pET32a-Trx-Alg2, pET28a-Alg11DTM, pET28a-Dpm1, pET28a-M-Alg3, pET28a-M-Alg9 and pET28a-Alg 12.
4. The method of claim 1 or 2, wherein: purifying the mannosyltransferase, wherein recombinant escherichia coli with prokaryotic expression of Alg1DTM, Trx-Alg2 and Dpm1 is respectively subjected to ultrasonic disruption, then respectively subjected to centrifugation to prepare Alg1DTM, Trx-Alg2 and Dpm1 membrane components, and the Alg1DTM, Trx-Alg2 and Dpm1 membrane components are respectively dissolved in 1% Triton X-100 buffer solution to obtain Alg1DTM, Trx-Alg2 and Dpm1 membrane proteins; carrying out ultrasonic crushing on recombinant escherichia coli of which the prokaryotic expression is Alg11DTM, and then centrifuging to obtain Alg11DTM supernatant protein; respectively purifying the Alg1DTM, Trx-Alg2 and Dpm1 membrane proteins by nickel ion affinity chromatography, and purifying the Alg11DTM supernatant protein by nickel ion affinity chromatography.
5. The method of claim 4, wherein: respectively preparing Alg1DTM, Trx-Alg2 and Dpm1 membrane components by centrifugation, wherein the recombinant escherichia coli of which the prokaryotic expression is Alg1DTM, Trx-Alg2 and Dpm1 is respectively subjected to ultrasonic disruption, centrifuged at 4000r/min for 20 min, deposited, collected supernatant, centrifuged at 15000r/min for 90 min at high speed, and discarded; and centrifuging to obtain Alg11DTM supernatant protein, wherein the centrifugation speed is 15000r/min, and the centrifugation time is 90 min.
6. The method of claim 1 or 2, wherein: the preparation method of the mannosyltransferase membrane component comprises the steps of respectively carrying out ultrasonic crushing on recombinant escherichia coli of which prokaryotic expression is expressed by M-Alg3, M-Alg9 and Alg12, centrifuging for 20 min at 4000r/min, discarding precipitates, collecting supernate, carrying out ultracentrifugation, discarding supernate, and obtaining the escherichia coli membrane component, wherein the ultracentrifugation speed is 100000r/min, and the time is 60 min.
7. The method of claim 1,2 or 5, wherein: synthesizing the donor substrate PP-Man, wherein the purified Dpm1 protein catalyzes phytyl-phosphate and donor GDP-Man to synthesize the PP-Man, and a reaction system of the PP-Man comprises the following components: 1 mM/muL Tris/HCl pH7.5, 0.2 mM/muL MgCl21% NP-40, 0.4 mM/. mu.L phytonyl-phosphate, 1 mM/. mu.L GDP-Man and 2 mg/mL Dpm1, wherein the reaction temperature is 30 ℃ and the reaction time is 10 hours.
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