Cell-free expression method of membrane-bound phosphate glycosyltransferase

Abstract

The invention discloses a cell-free expression method of membrane-bound glycosyl phosphate transferase. The cell-free expression method comprises the following steps: constructing a recombinant plasmid containing a coding membrane-bound glycosyl phosphate transferase gene GTp or epsL; preparing a nano liposome; the preparation method comprises the following steps: preparing an Escherichia coli Rosetta (DE3) cell extract; the method comprises the following steps: adding recombinant plasmids into a cell-free expression system containing an escherichia coli Rosetta (DE3) cell extract and a reaction buffer solution, supplementing a surfactant or nano-liposome as an artificial hydrophobic material into the cell-free expression system, uniformly mixing, incubating in a constant-temperature shaking table for 8-12 hours, and carrying out freeze-drying, thereby obtaining the cell-free expression system containing the escherichia coli Rosetta (DE3) cell extract. Therefore, the high-efficiency soluble expression of the GTp or epsL membrane combined phosphate glycosyl transferase is realized. The soluble expression of the membrane-bound glycosyl phosphate transferase GTp and epsL can be realized within several hours, so that the time cost for obtaining the membrane-bound glycosyl phosphate transferase is greatly reduced.

Description

Technical Field

[0001] The invention relates to the technical field of protein synthesis, and in particular to a cell-free expression method of membrane-bound phosphoglycosyltransferase. Background Art

[0002] Microbial exopolysaccharides not only contribute to the mutual adsorption and recognition between cells, stress response to extreme external environments, and adsorption of exogenous organic or inorganic compounds, but more importantly, the excellent rheological properties, emulsification and flocculation activity of exopolysaccharides give them special biological activities such as immunomodulation, anti-tumor, and antioxidant. Therefore, the important medical value and industrial use of microbial exopolysaccharides have attracted widespread attention.

[0003] The biosynthesis of microbial exopolysaccharides and glycoconjugates (glycolipids, glycoproteins) depends on phosphoglycosyltransferases to initiate the assembly of sugar repeating units or glycoconjugate precursors. Under the action of phosphoglycosyltransferases, sugar nucleotides and lipid carriers (Und-P) form Und-PP-sugar. Phosphoglycosyltransferases are divided into two families: polyisoprenyl phosphate N-acetylglucosamine-1-phosphotransferase (PNPT) and polyisoprenyl phosphate hexose-1-phosphotransferase (PHPT). However, since phosphoglycosyltransferase is an integral membrane protein, its hydrophobic domain makes it difficult to overexpress in organisms such as Escherichia coli. How to obtain membrane proteins with high purity and good stability is still a major challenge in the characterization of phosphoglycosyltransferases, which to some extent limits researchers' analysis of the biosynthesis mechanism of microbial exopolysaccharides and the regulation of sugar molecule composition.

[0004] The traditional expression method of membrane-bound phosphoglycosyltransferase is to express it in an intracellular expression system such as Escherichia coli by fusing molecular chaperones or lytic tags such as SUMO and TrxA (Protein Expression and Purification 207(2023)106273, Glycobiology, 2012, 22(1):116–122), and then use a series of surfactants such as DDM, Triton X-100, CHAPS to extract or reconstruct the insoluble protein precipitate expressed intracellularly from the membrane (Journal of Biological Chemistry, 2023, 299(10):105194, Antimicrobial Agents and Chemotherapy, 2017, 61(11):e01310-17, MBio, 2023, 14(5):e00948-23.), thereby dissolving it into detergent micelles to obtain soluble protein samples. Despite this, the functional study and crystal structure analysis of membrane-bound phosphoglycosyltransferases are still very difficult. This is mainly because overexpression of membrane proteins can easily cause cytotoxicity, and their expression level and purity have not yet reached the requirements for structural analysis; and not all micelles formed by surfactants can reconstruct phosphoglycosyltransferases in vitro or extract phosphoglycosyltransferases in vivo and make them have corresponding functional activity. This process still requires tedious screening and optimization of solubilizing tags or surfactants. Therefore, traditional research methods still have obvious limitations in achieving the functional expression of membrane-bound phosphoglycosyltransferases, and there is an urgent need to find an efficient method to achieve soluble expression of membrane-bound phosphoglycosyltransferases.

[0005] In recent years, with the progress of synthetic biology, the cell-free protein synthesis (CFPS) system based on cell extracts has become an open protein expression system that is expected to replace the intracellular system. With the exogenous supplementation of energy systems, amino acids, cofactors, gene templates and other necessary substances, the central dogma can be realized in vitro, gene transcription and protein translation can be controlled, and efficient synthesis of target proteins can be achieved within a few hours. Compared with the traditional intracellular expression system, CFPS is not only easy to operate and efficient, but also breaks through the bottleneck of the relationship between protein synthesis and cell activity, providing a new way for the functional expression of membrane proteins.

[0006] At present, surfactant-based cell-free (DCF) and lipid-based cell-free (LCF) models are used in CFPS systems to express membrane proteins, so that the target membrane protein can be co-translated with micelles or liposomes in a soluble form. The DCF model directly supplements surfactants when assembling the CFPS reaction system, and membrane proteins can use the micelles formed by them to stabilize their hydrophobic domains; while the LCF model supplements lipid membrane components such as nanoliposomes and nanophospholipid disks, so that the hydrophobic domains of membrane proteins are integrated into the phospholipid bilayer structure. Friederike Junge supplemented 0.5% Brij35 in the cell-free system and successfully obtained a soluble protein sample of endothelin receptor A in the DCF mode (Journal of Structural Biology, 2010, 172(1):94–106); Ke Yue prepared a liposome and achieved soluble expression of the water channel protein AqpZ in the LCF mode (Cells, 2019, 8(11):1325.); Michael C. Jewett's team used DDM, Triton X-100 and POPC nanophospholipid disks as artificial hydrophobic materials to obtain soluble oligosaccharyltransferase CjPglB in the CFPS system in the DCF or LCF mode (Biotechnology and Bioengineering, 2018, 115(3):739–750.).

[0007] However, there are few reports on the use of CFPS systems to prepare phosphoglycosyltransferases. This is mainly due to the lack of a biological membrane environment in the CFPS system, and the need to modify the membrane-enhanced system for the specific protein phosphoglycosyltransferase. In addition, the differences in the physicochemical properties of different membrane proteins lead to different optimal hydrophobic materials, and the high cost of commercial nanophospholipid disks limits the further application of this technology in the in vitro preparation of phosphoglycosyltransferases.

[0008] Therefore, it is crucial to screen suitable hydrophobic materials and establish a cell-free protein synthesis system suitable for the soluble expression of membrane-bound phosphoglycosyltransferases. Summary of the invention

[0009] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the purpose of the present invention is to provide a cell-free expression method of a membrane-bound phosphoglycosyltransferase, which solves the problems of low expression efficiency, poor solubility, and difficulty in folding into an active pocket series in the current synthesis process of membrane-bound phosphoglycosyltransferase.

[0010] In order to achieve the above object, the embodiment of the present invention provides a cell-free expression method of membrane-bound phosphoglycosyltransferase, which comprises the following steps:

[0011] Constructing a recombinant plasmid containing the gene encoding membrane-bound phosphoglycosyltransferase GTp or epsL;

[0012] preparing nanoliposomes;

[0013] Preparation of Escherichia coli Rosetta (DE3) cell extracts;

[0014] The recombinant plasmid is added to a cell-free expression system comprising the Escherichia coli Rosetta (DE3) cell extract and a reaction buffer, and a surfactant or nanoliposome is added to the cell-free expression system as an artificial hydrophobic material. After mixing, the mixture is incubated in a constant temperature shaker at 20 to 30° C. for 8 to 12 hours to achieve efficient soluble expression of GTp or epsL membrane-bound phosphoglycosyltransferase.

[0015] According to an embodiment of the present invention, a cell-free expression method of a membrane-bound phosphoglycosyltransferase is provided. A membrane-enhanced cell-free system based on Escherichia coli Rosetta (DE3) cell extract is used as an important protein synthesis platform. Soluble expression of membrane-bound phosphoglycosyltransferases GTp and epsL can be achieved in a 1.5 mL EP tube within a few hours, which greatly reduces the time cost of obtaining membrane-bound phosphoglycosyltransferases, simplifies their production process, and effectively avoids the problems of cumbersome expression process and low expression amount of traditional methods. It not only provides a new method for efficient soluble expression of membrane proteins, but also lays a foundation for crystal structure analysis and functional research of membrane-bound phosphoglycosyltransferases.

[0016] In addition, the cell-free expression method of a membrane-bound phosphoglycosyltransferase proposed in the above embodiment of the present invention may also have the following additional technical features:

[0017] Optionally, the amino acid sequence of membrane-bound phosphoglycosyltransferase GTp is shown as SEQ ID No.1; the amino acid sequence of membrane-bound phosphoglycosyltransferase epsL is shown as SEQ ID No.2.

[0018] Optionally, the steps of constructing a recombinant plasmid containing the gene encoding membrane-bound phosphoglycosyltransferase GTp or epsL are:

[0019] The genome of Bacillus licheniformis CGMCC 2876 was used as a template to amplify the membrane-bound phosphoglycosyltransferase gene GTp and the membrane-bound phosphoglycosyltransferase gene epsL by PCR. The PCR amplification products were connected with the linearized pIVEX 2.4c vector by Gibson assembly and transformed into Escherichia coli DH5α competent cells. The recombinants were screened and sequenced to obtain the pIVEX2.4c-GTp recombinant plasmid and the pIVEX2.4c-epsL recombinant plasmid.

[0020] Furthermore, the primers used for PCR amplification of the membrane-bound phosphoglycosyltransferase gene GTp are shown in SEQ ID No.3 and SEQ ID No.4; the primers used for PCR amplification of the membrane-bound phosphoglycosyltransferase gene epsL are shown in SEQ ID No.5 and SEQ ID No.6.

[0021] Optionally, the preparation of the nanoliposomes comprises:

[0022] Phospholipids and cholesterol were dissolved in an organic solvent and a lipid film was formed by rotary evaporation. After the organic solvent was removed, phosphate buffer was added and stirred for hydration at 45°C for 1 hour. After the lipid suspension was collected, the liposome particle size was immediately reduced by the combined action of ultrasonic energy and mechanical energy to obtain nanoliposomes.

[0023] Furthermore, the organic solvent is chloroform and methanol in a ratio of 3:1; the ratio of the phospholipid and the cholesterol is 4:1; and the amount of phosphate buffer added is: 1 mL of phosphate buffer for every 20 mg of phospholipid.

[0024] Furthermore, the combined action of ultrasonic energy and mechanical energy was as follows: using 20% ​​power, ultrasonication for 10 minutes and then immediately passing through a liposome extruder containing a 100-200 nm polycarbonate membrane 21-31 times.

[0025] Optionally, the components of the reaction buffer are HEPES, ATP, CTP, GMP, UMP, coenzyme A, tRNA, folinic acid, spermidine, potassium glutamate, magnesium glutamate, dithiothreitol, triphosphoglycerate, multiple amino acids, nicotinamide dinucleotide, and PEG6000.

[0026] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1The recombinant plasmids carrying the gene encoding membrane-bound phosphoglycosyltransferase according to Example 1 of the present invention are shown in Figure (a) for the pIVEX 2.4c-GTp plasmid map, and Figure (b) for the pIVEX 2.4c-epsL plasmid map;

[0028] Figure 2 This is an example of dynamic light scattering (DLS) analysis of nanoliposomes prepared according to Example 2 of the present invention;

[0029] Figure 3 This is an example of Western Blotting analysis of a 50 μL membrane-enhanced cell-free system expressing membrane-bound phosphoglycosyltransferase GTp in the DCF mode according to Example 3 of the present invention; in the figure, NC is a negative control, T is total protein, and S is soluble protein;

[0030] Figure 4 This is an example of Western Blotting analysis of expressing membrane-bound GTp in the DCF mode of Comparative Example 1 of the present invention, using DDM or Triton X-100 as an artificial hydrophobic material; in the figure, T is total protein and S is soluble protein;

[0031] Figure 5 This is an example of Western Blotting analysis of a 50 μL membrane-enhanced cell-free system expressing membrane-bound phosphoglycosyltransferase epsL in the DCF mode according to Example 4 of the present invention; in the figure, NC is a negative control, T is total protein, and S is soluble protein;

[0032] Figure 6 This is an example of Western Blotting analysis of expressing membrane-bound epsL using DDM or Triton X-100 as artificial hydrophobic material in the DCF mode of Comparative Example 2 of the present invention; in the figure, T is total protein and S is soluble protein;

[0033] Figure 7 This is an example of Western Blotting analysis of a 50 μL membrane-enhanced cell-free system expressing membrane-bound phosphoglycosyltransferase GTp in the LCF mode according to Example 5 of the present invention; in the figure, T is total protein and S is soluble protein;

[0034] Figure 8 This is an example of Western Blotting analysis of a 50 μL membrane-enhanced cell-free system expressing membrane-bound phosphoglycosyltransferase epsL in the LCF mode according to Example 6 of the present invention; in the figure, T is total protein and S is soluble protein. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0036] Unless otherwise defined, all technical and scientific terms in this application have the same meaning as commonly understood by those skilled in the art. If a definition used in this application conflicts or is inconsistent with a definition in other public documents, the definition used in this application shall prevail.

[0037] The term [cell-free expression] refers to the use of cell extracts, gene templates and reaction buffer to synthesize proteins in vitro.

[0038] The term [DCF mode] refers to cell-free expression using a surfactant-based mode (DCF), that is, a certain concentration of surfactant is added to the cell-free system before the reaction begins.

[0039] The term [LCF mode] refers to cell-free expression using a lipid based cell-free (LCF) mode, i.e., a certain concentration of lipid material is added to the cell-free system before the reaction begins.

[0040] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0041] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0042] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0043] Example 1 Construction of a recombinant plasmid encoding a phosphoglycosyltransferase gene

[0044] Using the genome of Bacillus licheniformis CGMCC 2876 (the Bacillus licheniformis was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee on January 14, 2009, and the deposit center number is CGMCC No. 2876) as a template, using the sequences shown in SEQ ID No. 3 and SEQ ID No. 4 as primers, the gene encoding GTp was amplified by PCR technology, and using the sequences shown in SEQ ID No. 5 and SEQ ID No. 6 as primers, the gene encoding epsL was amplified by PCR technology. Take 50-400ng genomic DNA, 25μL of 2x Phanta Max Master Mix polymerase (Vazyme), 2μL of 10μM upstream and downstream primers, and make up to 50μL with ddH2O. Then, PCR amplification was performed with the following procedure: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 50 s, 72°C complete extension for 5 min, and the whole system was cycled for 35 times. The amplified product was confirmed to be of the correct size by agarose gel electrophoresis, and then purified and recovered.

[0045] The in vitro expression vector pIVEX 2.4c was digested with XhoI and SmaI, and after purification and recovery, the PCR-recovered fragments of GTp and epsL were connected to the linearized vector pIVEX 2.4c using the Gibson assembly technique, and then transformed into Escherichia coli DH5α. After screening positive clones and sequencing verification, the pIVEX2.4c-GTp recombinant plasmid and pIVEX2.4c-epsL recombinant plasmid were extracted, as shown in Figure 1 All recombinant plasmids have been connected to the 6xHis-Tag sequence at the N-terminus of the target protein to facilitate subsequent detection of protein expression by western blotting.

[0046] Example 2 Preparation of Nanoliposomes

[0047] Method 1: Weigh 30 mg of dioleoylphosphatidylcholine (DOPC) and 7.5 mg of cholesterol and dissolve them in 8 mL of chloroform: methanol (3:1) solvent, form a lipid film by rotary evaporation, completely remove the organic solvent and add 1.5 mL of phosphate buffer, stir and hydrate at 45 ° C for 1 hour, collect the lipid suspension and use 20% power for 10 minutes, pass it through a liposome extruder containing a 200 nm polycarbonate membrane 31 times, so that the liposome particle size can be reduced by the combined action of ultrasonic energy (ultrasonic cell disruptor) and mechanical energy (liposome extruder); after dynamic light scattering characterization, such as Figure 2 As shown, the average particle size of DOPC nanoliposomes is about 185.36 nm, and the PDI value is 0.175.

[0048] Method 2: The same liposome preparation conditions as method 1 were used to investigate the effects of other methods of reducing particle size on the average particle size and PDI value of nanoliposomes. The difference is that the method used to reduce the particle size is: collecting the lipid suspension and using 20% ​​power for 10 to 20 minutes of ultrasound.

[0049] Method 3: The same liposome preparation conditions as method 1 were used to investigate the effects of other methods of reducing particle size on the average particle size and PDI value of nanoliposomes. The difference is that the method used to reduce the particle size is: collecting the lipid suspension and passing it through a liposome extruder containing a 200 nm polycarbonate membrane 21 to 31 times.

[0050] After dynamic light scattering characterization, the results are shown in Table 1. The liposome particle size was reduced by the combined action of ultrasonic energy (ultrasonic cell disruptor) and mechanical energy (liposome extruder), and the prepared nanoliposomes had better stability.

[0051] Table 1: Results of the average particle size and PDI value of nanoliposomes

[0052] Average particle size (nm) PDI value Method 1 185.36 0.175 Method 2 189.60 0.412 Method 3 190.82 0.306

[0053] Example 3: Using 50 μL of membrane-enhanced cell-free system to express membrane-bound GTp in DCF mode

[0054] 1. Preparation of cell extract: Inoculate a single colony of Escherichia coli Rosetta (DE3) after activation into 2xYTP medium and culture overnight at 30°C; 600 The inoculum of about 0.05 was transferred to 2xYTP secondary seed solution and cultured at 37°C until OD 600 When the OD value was 0.6 to 0.8, 1 mM IPTG was used to induce the expression of T7 RNA polymerase and the culture was continued until the OD value was 600 After about 3, the cells were collected and the cell pellet was washed 3 times with pre-cooled S30A buffer (14mM magnesium glutamate; 60mM potassium glutamate; 50mM Tris, 2mM DTT, pH7.7). Each 1g of wet cells was resuspended in 1mL S30A buffer and ultrasonically disrupted. 650J of ultrasonic energy was input for each 1mL of resuspension to obtain cell lysate. The lysate was centrifuged at 12,000×g for 10 minutes at 4°C, the supernatant was taken, and 2mM dithiothreitol (DTT) was added. The sample was incubated at 37°C for 80 minutes, centrifuged at 12,000×g for 10 minutes at 4°C, the supernatant was taken, flash frozen in liquid nitrogen, and stored at -80°C for use.

[0055] 2. 2.5X reaction buffer configuration: 158mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 4.73mM ATP, 4.73mM CTP, 3.92mM GMP, 4.5mM UMP, 0.8mM coenzyme A, 0.63mg / mL tRNA, 0.2mM folinic acid, 3.15mM spermidine, 450mM potassium glutamate, 50mM magnesium glutamate, 1.25mM dithiothreitol, 85mM triphosphoglycerate, 7.5mM 20 kinds of amino acids (arginine, valine, tryptophan, phenylalanine, isoleucine, leucine, cysteine, methionine, alanine, asparagine, glycine, glutamine, glycine, histidine, lysine, proline, serine, glutamic acid, threonine, tyrosine), 1mM nicotinamide dinucleotide, 5% PEG6000.

[0056] 3. Cell-free expression of GTp: In a 1.5 mL EP tube, add 15 μL of Escherichia coli Rosetta (DE3) extract (obtained in step 1), 750 ng of pIVEX2.4c-GTp recombinant plasmid (obtained in Example 1), 20 μL of 2.5X reaction buffer (obtained in step 2) and 2.5 μL of 20% Brij78 (polyoxyethylene stearate) mother solution in sequence, and use DEPC water to make up the reaction volume to 50 μL. After mixing the reaction components, place them in a constant temperature shaker at 24-30°C and 220 rpm for 8-12 hours of protein synthesis. After the reaction is completed, centrifuge the cell-free reaction solution in a refrigerated centrifuge at 4°C and 12,000×g for 20 minutes, and take the supernatant for Western blotting protein identification. The results are as follows. Figure 3 As shown, the membrane-enhanced cell-free system supplemented with a final concentration of 1% Brij78 effectively increased the soluble expression level of membrane-bound GTp.

[0057] Comparative Example 1: In DCF mode, DDM or Triton X-100 was used as an artificial hydrophobic material to express membrane-bound GTp

[0058] The same cell-free reaction conditions as in Example 3 were used to examine the applicability of surfactants suitable for soluble expression of other membrane-bound proteins (oligosaccharyl transferase CjPglB) in the DCF mode to membrane-bound GTp. The difference was that the surfactants used were DDM or Triton X-100. The results were as follows: Figure 4 As shown, no soluble GTp was produced and DDM or Triton X-100 was not suitable for cell-free soluble expression of the membrane-bound phosphoglycosyltransferase GTp.

[0059] Example 4: Taking 50 μL of membrane-enhanced cell-free system expressing membrane-bound epsL in DCF mode as an example

[0060] The cell-free reaction conditions were the same as those in Example 3 to investigate the applicability of the cell-free system to other membrane-bound phosphoglycosyltransferases, except that the recombinant plasmid used was pIVEX2.4c-epsL (obtained in Example 1).

[0061] The results are as follows Figure 5 As shown, the membrane-enhanced cell-free system supplemented with 1% Brij78 at a final concentration effectively increased the soluble expression level of membrane-bound epsL.

[0062] Comparative Example 2: In DCF mode, DDM or Triton X-100 was used as the artificial hydrophobic material to express membrane-bound epsL

[0063] The same cell-free reaction conditions as in Example 3 were used to examine the applicability of surfactants suitable for soluble expression of other membrane-bound proteins (oligosaccharyl transferase CjPglB) in the DCF mode to membrane-bound epsL. The difference was that the surfactants used were DDM or Triton X-100. The results were as follows: Figure 6 As shown, no soluble epsL was produced and DDM or Triton X-100 was not suitable for cell-free soluble expression of the membrane-bound phosphoglycosyltransferase epsL.

[0064] Example 5: Expression of membrane-bound GTp in a 50 μL membrane-enhanced cell-free system in LCF mode

[0065] The effect of other hydrophobic materials on improving the solubility of membrane proteins was investigated under the same cell-free reaction conditions as in Example 3. The difference was that the hydrophobic material used was 2.8 mg / mL DOPC nanoliposomes (obtained in Example 2).

[0066] The results are as follows Figure 7 As shown, the membrane-enhanced cell-free system supplemented with 2.8 mg / mL DOPC nanoliposomes effectively increased the soluble expression level of membrane-bound GTp.

[0067] Example 6: Using 50 μL of membrane-enhanced cell-free system to express membrane-bound epsL in LCF mode

[0068] The effect of other hydrophobic materials on improving the solubility of membrane proteins was investigated under the same cell-free reaction conditions as in Example 4. The difference was that the hydrophobic material used was 1.6 mg / mL DOPC nanoliposomes (obtained in Example 2).

[0069] The results are as follows Figure 8 As shown, the membrane-enhanced cell-free system supplemented with 1.6 mg / mL DOPC nanoliposomes effectively increased the soluble expression level of membrane-bound epsL.

[0070] In summary, according to the embodiments of the present invention, a membrane-enhanced cell-free system based on Escherichia coli Rosetta (DE3) cell extract is used as an important protein synthesis platform, and the soluble expression of membrane-bound phosphoglycosyltransferases GTp and epsL can be achieved in a 1.5 mL EP tube within a few hours, which greatly reduces the time cost of obtaining membrane-bound phosphoglycosyltransferases, simplifies their production process, and effectively avoids the problems of cumbersome expression process and low expression amount of traditional methods. It not only provides a new method for efficient soluble expression of membrane proteins, but also lays the foundation for crystal structure analysis and functional research of membrane-bound phosphoglycosyltransferases.

[0071] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0072] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A cell-free expression method for a membrane-bound phosphoglycosyltransferase, characterized in that: The following steps are involved: Constructing a recombinant plasmid containing the gene encoding membrane-bound phosphoglycosyltransferase GTp or epsL; preparing nanoliposomes; Preparation of Escherichia coli Rosetta (DE3) cell extracts; The recombinant plasmid is added to a cell-free expression system comprising the Escherichia coli Rosetta (DE3) cell extract and a reaction buffer, and a surfactant or nanoliposome is added to the cell-free expression system as an artificial hydrophobic material. After mixing, the mixture is incubated in a constant temperature shaker at 20 to 30° C. for 8 to 12 hours to achieve efficient soluble expression of GTp or epsL membrane-bound phosphoglycosyltransferase.

2. The cell-free expression method of membrane-bound phosphoglycosyltransferase according to claim 1, characterized in that: The amino acid sequence of membrane-bound phosphoglycosyltransferase GTp is shown in SEQ ID No.1; the amino acid sequence of membrane-bound phosphoglycosyltransferase epsL is shown in SEQ ID No.

2.

3. The cell-free expression method of membrane-bound phosphoglycosyltransferase according to claim 1, characterized in that: The steps for constructing a recombinant plasmid containing the gene encoding membrane-bound phosphoglycosyltransferase GTp or epsL are as follows: The genome of Bacillus licheniformis CGMCC 2876 was used as a template to amplify the membrane-bound phosphoglycosyltransferase gene GTp and the membrane-bound phosphoglycosyltransferase gene epsL by PCR. The PCR amplification products were connected with the linearized pIVEX 2.4c vector by Gibson assembly and transformed into Escherichia coli DH5α competent cells. The recombinants were screened and sequenced to obtain the pIVEX2.4c-GTp recombinant plasmid and the pIVEX2.4c-epsL recombinant plasmid.

4. The cell-free expression method of membrane-bound phosphoglycosyltransferase according to claim 3, characterized in that: The primers used for PCR amplification of the membrane-bound phosphoglycosyltransferase gene GTp are shown in SEQ ID No. 3 and SEQ ID No. 4; The primers used for PCR amplification of the membrane-bound phosphoglycosyltransferase gene epsL are shown in SEQ ID No.5 and SEQ ID No.

6.

5. The cell-free expression method of membrane-bound phosphoglycosyltransferase according to claim 1, characterized in that: The preparation of the nanoliposome comprises: Phospholipids and cholesterol were dissolved in an organic solvent and a lipid film was formed by rotary evaporation. After the organic solvent was removed, phosphate buffer was added and stirred for hydration at 45°C for 1 hour. After the lipid suspension was collected, the liposome particle size was immediately reduced by the combined action of ultrasonic energy and mechanical energy to obtain nanoliposomes.

6. The cell-free expression method of membrane-bound phosphoglycosyltransferase according to claim 5, characterized in that: The organic solvent is chloroform and methanol in a ratio of 3:1; the ratio of the phospholipid to the cholesterol is 4:1; and the amount of phosphate buffer added is: 1 mL of phosphate buffer is added for every 20 mg of phospholipid.

7. The cell-free expression method of membrane-bound phosphoglycosyltransferase according to claim 5, characterized in that: The combined effect of ultrasonic energy and mechanical energy is as follows: using 20% ​​power, ultrasonication for 10 minutes and immediately passing through a liposome extruder containing a 100-200 nm polycarbonate membrane 21-31 times.

8. The cell-free expression method of membrane-bound phosphoglycosyltransferase according to claim 1, characterized in that: The components of the reaction buffer are HEPES, ATP, CTP, GMP, UMP, coenzyme A, tRNA, folinic acid, spermidine, potassium glutamate, magnesium glutamate, dithiothreitol, triphosphoglyceric acid, multiple amino acids, nicotinamide dinucleotide, and PEG6000.

9. The cell-free expression method of membrane-bound phosphoglycosyltransferase according to claim 1, characterized in that: The surfactant is Brij78; the final concentration of the surfactant is 1.0-1.5%; the concentration of the nanoliposome is 1.2-3.2 mg / mL.

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