Recombinant chondroitin 6-O-sulfotransferase truncation and application thereof in synthesis of chondroitin sulfate C

Abstract

The invention discloses a recombinant chondroitin 6-O-sulfotransferase truncation and application thereof in synthesis of chondroitin sulfate C, and belongs to the technical field of bioengineering. On the basis of chondroitin 6-O-sulfotransferase from Norway rats, a chondroitin 6-O-sulfotransferase truncation body is obtained by truncating first 11-106 amino acids at the N end of RnCHST3, the sulfonation rate higher than that of the original RnCHST3 is shown, the sulfonation rate is increased by 51%, and the sulfonation rate in 24 h can reach 11.5%. On the basis of the delta 106RnCHST3, the pET28a (+)-MBP delta 106RnCHST3 for MBP fusion expression is constructed, and the soluble expression quantity of the pET28a (+)-MBP delta 106RnCHST3 is remarkably improved. According to the invention, the chondroitin 6-O-sulfotransferase truncation is used for catalytic preparation of CSC, and a novel green and environment-friendly method is provided.

Description

Technical Field

[0001] The invention relates to a recombinant chondroitin 6-O-sulfotransferase truncation and application thereof in synthesizing chondroitin sulfate C, belonging to the technical field of bioengineering. Background Art

[0002] Chondroitin Sulfate C (CSC), the disaccharide unit is GlcAβ1-3GalNAc (6S), and its structure is a sulfate group modification on the C-6 hydroxyl position of N-acetylgalactosamine in the chondroitin skeleton. Chondroitin is a linear polysaccharide composed of disaccharide units formed by glucuronic acid and N-acetylgalactosamine. Chondroitin sulfate is widely used in the medical field, including bone and joint repair, neural development, cancer treatment and other fields. When used in combination with glucosamine, it has the effects of relieving pain and promoting cartilage regeneration; it can improve the activity of the central nervous system and is beneficial to the development of the brain; it participates in cancer treatment as a targeting molecule through interaction with the multifunctional cell surface adhesion receptor (CD44).

[0003] CSC synthesis can be divided into three categories: tissue extraction, chemical synthesis, and biosynthesis. (1) Tissue extraction method: In industry, CSC is extracted from tissues and organs as raw materials, which requires complex and lengthy steps. This process has problems such as complex steps, high energy consumption, and easy introduction of viruses. (2) Chemical synthesis method: Hsieh-Wilson et al. reported a synthesis method for CSC tetrasaccharide, but there are problems such as complex reaction steps and harsh reaction conditions. (3) Biosynthesis method: Kang Zhen et al. expressed chondroitin 6-O-sulfotransferase through Pichia pastoris GS115 recombinant bacteria and secreted it into the culture medium. The enzyme method catalyzed the synthesis of CSC from chondroitin, and the enzyme activity was 0.20±0.01U / mg when chondroitin was used as a substrate. However, the activity of sulfotransferase was low and the expression was poor. Therefore, it is urgent to develop new methods to improve the sulfonation rate and soluble expression of chondroitin 6-O-sulfotransferase. Summary of the invention

[0004] The present invention provides a chondroitin 6-O-sulfotransferase truncation, which is obtained by truncating the amino acids at positions 1 to 11, 1 to 14, 1 to 58, 1 to 62, 1 to 65, 1 to 73, 1 to 106 or 1 to 131 of the N-terminus of the chondroitin 6-O-sulfotransferase RnCHST3 whose amino acid sequence is shown in SEQ ID NO.1.

[0005] In one embodiment, the truncation is any one of the following (a) to (h):

[0006] (a) truncating the amino acids 1 to 11 at the N-terminus of the chondroitin 6-O-sulfotransferase RnCHST3 shown in the amino acid sequence of SEQ ID NO.1 to obtain a truncated product shown in SEQ ID NO.2, named Δ11RnCHST3;

[0007] (b) truncating the amino acids 1 to 14 at the N-terminus of the chondroitin 6-O-sulfotransferase RnCHST3 shown in the amino acid sequence of SEQ ID NO.1 to obtain a truncated product shown in SEQ ID NO.3, named Δ14RnCHST3;

[0008] (c) truncating the amino acids 1 to 58 at the N-terminus of the chondroitin 6-O-sulfotransferase RnCHST3 shown in the amino acid sequence of SEQ ID NO.1 to obtain a truncated product shown in SEQ ID NO.4, named Δ58RnCHST3;

[0009] (d) truncating the amino acids 1 to 11 at the N-terminus of the chondroitin 6-O-sulfotransferase RnCHST3 shown in the amino acid sequence of SEQ ID NO.1 to obtain a truncated product shown in SEQ ID NO.5 and named Δ62RnCHST3;

[0010] (e) truncating the amino acids 1 to 11 at the N-terminus of the chondroitin 6-O-sulfotransferase RnCHST3 with the amino acid sequence shown in SEQ ID NO.1 to obtain a truncated product shown in SEQ ID NO.6, named Δ65RnCHST3;

[0011] (f) truncating the amino acids 1 to 11 at the N-terminus of the chondroitin 6-O-sulfotransferase RnCHST3 shown in the amino acid sequence of SEQ ID NO.1 to obtain a truncated product shown in SEQ ID NO.7, named Δ73RnCHST3;

[0012] (g) truncating the amino acids 1 to 11 at the N-terminus of the chondroitin 6-O-sulfotransferase RnCHST3 shown in the amino acid sequence of SEQ ID NO.1 to obtain a truncated product shown in SEQ ID NO.8 and named Δ106RnCHST3;

[0013] (h) The amino acid sequence of chondroitin 6-O-sulfotransferase RnCHST3 shown in SEQ ID NO.1 was truncated at positions 1 to 11 at the N-terminus to obtain a truncated product shown in SEQ ID NO.9 and named Δ131RnCHST3.

[0014] In one embodiment, the N-terminus of the chondroitin 6-O-sulfotransferase truncation is further connected to a solubility-promoting tag.

[0015] In one embodiment, the lytic tag includes but is not limited to 12×His, TrxA or MBP.

[0016] In one embodiment, the amino acid sequence of the lytic tag 12×His is shown as SEQ ID NO.14; the amino acid sequence of the lytic tag TrxA is shown as SEQ ID NO.15; and the amino acid sequence of the lytic tag MBP is shown as SEQ ID NO.16.

[0017] The present invention also provides a gene encoding the chondroitin 6-O-sulfotransferase truncation.

[0018] The invention also provides a recombinant vector carrying the gene.

[0019] In one embodiment, the recombinant vector uses pET-28a as an expression vector.

[0020] The present invention also provides a microbial cell carrying the gene or the recombinant vector.

[0021] In one embodiment, the microbial cell is a prokaryotic or eukaryotic expression host.

[0022] In one embodiment, the microbial cell uses Escherichia coli Rosetta (DE3) as an expression host.

[0023] The present invention also provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli expresses the chondroitin 6-O-sulfotransferase truncation.

[0024] In one embodiment, the recombinant Escherichia coli uses Escherichia coli Rosetta (DE3) as an expression host and pET-28a as an expression vector.

[0025] The present invention also provides a method for obtaining the above-mentioned recombinant chondroitin 6-O-sulfotransferase RnCHST3 truncation, the method comprising the following steps:

[0026] (1) Determining the truncated amino acid based on the amino acid sequence of the recombinant chondroitin 6-O-sulfotransferase RnCHST3; designing truncation primers, using the vector carrying the recombinant chondroitin 6-O-sulfotransferase RnCHST3 gene as a template for truncation; constructing a plasmid vector containing the truncation;

[0027] (2) transforming the plasmid vector containing the truncation into a host cell;

[0028] (3) Select positive clones for fermentation culture and purify chondroitin 6-O-sulfotransferase RnCHST3.

[0029] In one embodiment of the present invention, the host cell is Escherichia coli.

[0030] The present invention also provides a method for increasing the expression level of mouse chondroitin 6-O-sulfotransferase, the method comprising (a), (b) or (c):

[0031] (a) truncating the amino acid sequence of the chondroitin 6-O-sulfotransferase; the truncation is to truncate the amino acids at positions 1 to 11, 1 to 14, 1 to 58, 1 to 62, 1 to 65, 1 to 73, 1 to 106 or 1 to 131 of the N-terminus of the chondroitin 6-O-sulfotransferase;

[0032] (b) connecting a solubility-promoting tag 12×Hi, TrxA or MBP to the N-terminus of the chondroitin 6-O-sulfotransferase;

[0033] (c) truncating the amino acid sequence of the chondroitin 6-O-sulfotransferase; the truncation is to truncate the amino acids 1 to 11, 1 to 14, 1 to 58, 1 to 62, 1 to 65, 1 to 73, 1 to 106 or 1 to 131 at the N-terminus of the chondroitin 6-O-sulfotransferase, and connecting a solubilizing tag 12×Hi, TrxA or MBP to the N-terminus of the truncated chondroitin 6-O-sulfotransferase.

[0034] In one embodiment, the amino acid sequence of the chondroitin 6-O-sulfotransferase is shown in SEQ ID NO.1

[0035] The present invention also provides application of the recombinant Escherichia coli or the method in producing chondroitin sulfate or a product containing chondroitin sulfate.

[0036] The present invention also provides a method for preparing chondroitin sulfate C, which comprises reacting the recombinant chondroitin 6-O-sulfotransferase RnCHST3 truncation, or the microbial cell, or the crude enzyme or pure enzyme of the recombinant Escherichia coli in a reaction system containing chondroitin and 3'-adenosine phosphate-5'-phosphosulfate.

[0037] In one embodiment, the microbial cells or the recombinant Escherichia coli are added to the reaction system at a final concentration of 10 to 40 g / L.

[0038] In one embodiment, the final concentration of chondroitin in the reaction system is 5-20 g / L.

[0039] In one embodiment, the final concentration of the 3'-phosphoadenosine-5'-phosphosulfate in the reaction system is 5.1-20.4 g / L.

[0040] In one embodiment, the reaction conditions are: pH 6.5-8.5, 37° C., for 24-48 hours.

[0041] The present invention also provides the use of the recombinant chondroitin 6-O-sulfotransferase RnCHST3 truncation, or the gene, or the recombinant vector, or the recombinant cell, or the recombinant Escherichia coli in preparing a product containing chondroitin sulfate C.

[0042] Beneficial effects:

[0043] (1) The present invention provides a truncation of chondroitin 6-O-sulfotransferase RnCHST3, the conversion rate of chondroitin 6-O sulfonation to chondroitin sulfate C of the truncation is increased by 51% compared with the recombinant chondroitin 6-O-sulfotransferase RnCHST3, and while the sulfonation rate is increased, the soluble expression is also improved.

[0044] (2) The chondroitin 6-O-sulfotransferase RnCHST3 truncation constructed by the present invention exhibits higher chondroitin 6-O sulfonation activity. The sulfonation rate reaches 11.5% under the conditions of 37° C. and pH 7.5 for 24 hours.

[0045] (3) The present invention also utilizes a method of fusion expression of a solubility-promoting tag to significantly increase the soluble expression level of the truncation. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the reaction catalyzed by chondroitin 6-O-sulfotransferase.

[0047] Figure 2 Protein expression analysis of 8 different RnCHST3 truncations.

[0048] Figure 3 LC-MS product identification of RnCHST3-catalyzed chondroitin 6-O-sulfonation to CSC.

[0049] Figure 4 The sulfonic acid conversion rates of 8 different RnCHST3 truncations are shown.

[0050] Figure 5 Analysis of the expression of the fusion protein of the solubility tag and Δ106RnCHST3. DETAILED DESCRIPTION

[0051] Reagents and materials:

[0052] Prime STAR Max DNA polymerase, BamHI, HindIII, NdeI and XhoI endonucleases, DNA Marker and other enzyme reagents were purchased from TaKaRa (Dalian).

[0053] ClonExpress one-step directional cloning kit and gel recovery kit were purchased from Vazyme Biotech (Nanjing).

[0054] The plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0055] Various analytical reagents were purchased from Sinopharm Group.

[0056] Culture medium:

[0057] LB medium (g / L): 5% yeast powder, 10% NaCl, and 10% peptone; dilute to the corresponding volume with deionized water, sterilize at 121°C for 20 min and set aside; about 2% agar powder should be added to the solid medium before sterilization.

[0058] TB culture medium (g / L): 4 g of glycerol, 24 g of yeast powder (Angel 802), 12 g of tryptone, 2.31 g of KH2PO4, and 16.42 g of K2HPO4·3H2O. Add deionized water to make up to the corresponding volume, sterilize at 121°C for 15-20 min, and set aside for use.

[0059] In vitro catalytic reaction of chondroitin sulfate 6-O-sulfotransferase:

[0060] Preparation of reaction solution: Use 20mmol / L Tris pH 7.5 prepared at 37°C as the reaction solvent, add crude enzyme solution prepared by crushing the bacteria with a final concentration of 20g / L, control the final concentration of dissolved chondroitin in the reaction solution to be 15g / L, and the final concentration of dissolved 3'-phosphoadenosine-5'-phosphosulfate to be 15.3g / L.

[0061] CSC detection method:

[0062] The sulfonation efficiency of CSC is defined as the ratio of the content of CSC disaccharide (Di-6S) to the total amount of Di-6S and chondroitin disaccharide (Di-0S). HPLC separation and detection were used to perform quantitative analysis by calculating the peak area ratio of Di-6S and Di-0S. Take 1 mL of fermentation broth, add 20 μL of 20 mg / mL chondroitin lyase ABC I, incubate at 37 ° C for 12 h to completely cleave it into disaccharides, inactivate the protein in a boiling water bath for 10 min, centrifuge at 12,000 × g for 20 min, remove impurities through a MW3000 ultrafiltration tube, and the purified disaccharides are filtered through a 0.22 μm filter membrane and then tested by HPLC.

[0063] HPLC detection conditions are:

[0064] A Pro Pac SAX-10 chromatographic column (specification: 4.0×250mm) was used, mobile phase A: 10mmol / L Tris (pH adjusted to 3.5 with dilute hydrochloric acid), mobile phase B: 10mmol / L Tris + 1mol / L sodium chloride solution (pH adjusted to 3.5 with dilute hydrochloric acid), flow rate was 1.0mL / min, detector was an ultraviolet detector, detection wavelength: 232nm, column temperature: 40℃, injection volume: 10μL, and gradient elution was adopted.

[0065] Table 1 Enzymatic hydrolysis liquid phase gradient elution table

[0066]

[0067] Calculation of sulfonation rate: The sulfonation efficiency of CSC was calculated according to the ratio of the content of CSC disaccharide (Di-6S) to the total amount of Di-6S and chondroitin disaccharide (Di-0S).

[0068] Example 1: Construction and verification of RnCHST3 expression strain

[0069] The GenBank accession number EDL93054.1 (amino acid sequence shown in SEQ ID NO.1) was submitted to Suzhou Jinweizhi Biotechnology Co., Ltd. for codon optimization and artificial synthesis. The synthesized nucleotide sequence was connected and assembled between the BamH I and Hind III restriction sites on the pET28a(+) plasmid to obtain the pET28a(+)-RnCHST3 plasmid, which was transferred into commercial E. coli Rosetta (DE3) to obtain a recombinant strain named E. coliRosetta(DE3)-pET28a(+)-RnCHST3.

[0070] Example 2: Analysis of protein expression of RnCHST3

[0071] A single colony of E. coli Rosetta (DE3)-RnCHST3 constructed in Example 1 was inoculated into 30 mL of fresh LB medium containing Kan and Cm resistance, and cultured at 37°C with shaking for 10 h. The inoculum was transferred to 150 mL of TB medium (containing Kan and Cm resistance) at a 1% (v / v) inoculum, and cultured at 37°C and 200 rpm until OD 600The p-value was 0.6-0.8, and IPTG was added at a final concentration of 60 mg / L for induction. The induction temperature was 16°C and the induction time was 18h. After the induction, centrifuge at 4°C and 8000rpm for 5min, wash the bacteria several times with PBS buffer, resuspend at 10mg / mL, and ultrasonically disrupt the bacteria for 10min (power 100W, ultrasound 2s, interval 1.5s), high-speed refrigerated centrifugation 4°C, 12000rpm, 10min. Taking out the supernatant is the supernatant sampling. Use a 1mL pipette tip to absorb 1mL PBS and resuspend, 4°C, 12000rpm, 2min (repeat twice), and take a sample (precipitation). Add 10μL 4×SDS loading to the supernatant precipitate, boil it in a pot for 10min, and run protein gel. The protein expression of RnCHST3 is shown in the figure. Figure 2 .

[0072] Example 3: Construction and verification of eight truncated expression strains of RnCHST3

[0073] Using the pET28a(+)-RnCHST3 constructed in Example 1 as a template, primers for truncation of Δ11RnCHST3, Δ14RnCHST3, Δ58RnCHST3, Δ62RnCHST3, Δ65RnCHST3, Δ73RnCHST3, Δ106RnCHST3, and Δ131RnCHST3 with 11, 14, 58, 62, 65, 73, 106, and 131 amino acids truncated from the N-terminus were designed, as shown in Table 2, and the truncation was constructed by whole plasmid PCR.

[0074] Table 2 Truncated primer sequence list

[0075]

[0076] Construct the PCR amplification system: 25 μL Prime STAR Max DNA polymerase, 1 μL each of the two primers for each truncation, 1 μL template (RnCHST3), 22 μL water; PCR reaction conditions are: ① 94℃ 3 min; ② 98℃ 10 s; ③ 58℃ 5 s; ④ 72℃ 10 s / kb; ⑤ cycle ② to ④ for 29 times; ⑥ 72℃ 5 min; ⑦ keep warm at 12℃. The above reaction system was incubated at 37°C for 1 hour to digest the plasmid template (the digestion system was: 1 μL DpnI, 44 μL PCR product of the above reaction, and 5 μL 10×T Buffer). After the digestion, the digestion product obtained was introduced into E. coli Rosetta competent cells by chemical transformation. The specific steps of the chemical transformation method were as follows: (1) 10 μL homologous recombination product was introduced into 100 μL E. coli Rosetta (DE3) competent cells; (2) ice bath for 10 minutes; (3) heat shock at 42°C water bath for 1 minute 30 seconds, and then quickly put into ice and stand in an ice bath for 4 minutes; (4) 600 μL non-resistant LB medium was added and mixed, and cultured at 37°C, 200 rpm for 1 hour; (5) centrifuged at 5000 rpm for 2 minutes to collect the bacteria; (6) the supernatant was removed, and the remaining 100-200 μL was mixed by pipetting and coated on an LB resistance plate containing 0.05 mg / mL kanamycin, and cultured at 37°C for about 12 hours. (7) Pick a single clone and place it in LB containing 0.05 mg / mL kanamycin resistance. After culturing at 200 rpm and 37°C for 12 h, send it to the company for sequencing. The one with correct sequencing is the positive transformant.

[0077] Genetically engineered bacteria E.coli Rosetta(DE3)-pET28a(+)-Δ11RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ14RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ58RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ58RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ62RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ65RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ73RnCHST3, E.coliRosetta(DE3)-pET28a(+)-Δ106RnCHS T3, E. coli Rosetta(DE3)-pET28a(+)-Δ131RnCHST3.

[0078] Example 4: Analysis of protein expression of 8 truncations

[0079] Pick out the E.coli Rosetta(DE3)-pET28a(+)-Δ11RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ14RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ58RnCHST3, and E.coli Rosetta(DE3)-pET28a(+)-Δ11RnCHST3, respectively) Rosetta(DE3)-pET28a(+)-Δ62RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ65RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ73RnCHST3, E.coli Rosetta(DE3)-pET28a(+)-Δ106RnCHST3, E.coli A single colony of Rosetta(DE3)-pET28a(+)-Δ131RnCHST3 was inoculated into 30 mL of fresh LB medium containing Kan and Cm resistance and cultured at 37°C with shaking for 10 h. The inoculum was transferred to 150 mL of TB medium (containing Kan and Cm resistance) at a 1% (v / v) inoculation volume and cultured at 37°C and 200 rpm until OD 600 The p-value was 0.6-0.8, and IPTG with a final concentration of 60 mg / L was added for induction. The induction temperature was 16°C and the induction time was 18h. After the induction, centrifuge at 4°C and 8000rpm for 5min, wash the bacteria several times with PBS buffer, resuspend at 10mg / mL, and ultrasonically disrupt the bacteria for 10min (power 100W, ultrasound 2s, interval 1.5s), high-speed refrigerated centrifugation 4°C, 12000rpm, 10min. Taking out the supernatant is the supernatant sampling. Use a 1mL pipette tip to absorb 1mL PBS and resuspend, 4°C, 12000rpm, 2min (repeat twice), and take a sample (precipitation). Add 10μL 4×SDS loading to the supernatant precipitate, place it in a pot and boil it for 10min, and run protein gel. The protein expression of 8 truncations, such as Figure 2 shown.

[0080] Example 5: Enzymatic conversion experiment of chondroitin 6-O sulfonation and product identification

[0081] The reaction used a 20mM Tris-HCl pH 7.5 system: 20g / L of bacteria was broken into a crude enzyme solution and added to the reaction system, and chondroitin with a final concentration of 15g / L and 3'-adenosine phosphate-5'-phosphosulfate with a final concentration of 15.3g / L were added. The catalytic reaction was carried out at 37°C for 24h. Take 1mL of fermentation broth, add 20μL of 20g / L chondroitin lyase ABC I (sigma-aldrich; product number: C2905), incubate at 37°C for 12h to completely cleave it into disaccharides, inactivate the protein in a boiling water bath for 10min, and centrifuge at 12,000×g for 20min. After impurities were removed by ultrafiltration tube of MW3000, the purified disaccharides were filtered through a 0.22μm filter membrane and then detected by HPLC. Liquid chromatography-mass spectrometry further proved the generation of the product CSC, such as Figure 3 shown.

[0082] Example 6: Conversion experiment of 8 truncated chondroitin 6-O sulfonation

[0083] In the 20mM Tris-HCl pH 7.5 system reaction, chondroitin with a final concentration of 15g / L and APS with a final concentration of 15.3g / LP were added. At 37°C, the crude enzyme solution after the bacterial cell crushing with a final concentration of 20g / L was used to catalyze the reaction for 24h. Take 1mL of fermentation broth, add 20μL of 20g / L chondroitin lyase ABC I, incubate at 37°C for 12h to completely cleave it into disaccharides, inactivate the protein in a boiling water bath for 10min, and centrifuge at 12,000×g for 20min. Then, impurities were removed by ultrafiltration tube of MW3000, and the purified disaccharides were filtered through a 0.22μm filter membrane and then detected by HPLC. The conversion of chondroitin 6-O sulfonation catalyzed by 8 truncated forms, such as Figure 4 shown.

[0084] Example 7: Optimization of the soluble expression level of Δ106RnCHST by fusion expression of a solubility-promoting tag

[0085] Using the nucleotide sequence of 12×His shown in SEQ ID NO.11, the nucleotide sequence of TrxA shown in SEQ ID NO.12 and the MBP shown in SEQ ID NO.13 as solubility tags, primers of pET28a(+)-12×His_Δ106RnCHST3, pET28a(+)-TrxA_Δ106RnCHST3 and pET28a(+)-MBP_Δ106RnCHST3 were designed, as shown in Table 3.

[0086] Table 3 Solubility-promoting tag primer sequence list

[0087]

[0088] The reaction PCR amplification system was constructed: 25 μL of Prime STAR Max DNA polymerase, 1 μL of each of the two primers of each truncation, 1 μL of template (RnCHST3), and 22 μL of water; the PCR reaction conditions were: ①94℃3min; ②98℃10s; ③58℃5s; ④72℃10s / kb; ⑤29 cycles of the three steps ② to ④; ⑥72℃5min; ⑦12℃ insulation. The above PCR reaction solutions were gel-recovered respectively. Then, the one-step homologous recombination kit was used to integrate them into the pET28a(+)-Δ106RnCHST3 plasmid, and the fusion tag construction position was located at the N-terminus of Δ1061RnCHST3, and co-expressed as a fusion protein. The chemical transformation method was used to introduce the E. coli Rosetta competent cells. The specific steps of the chemical transformation method are as follows: (1) 10 μL of homologous recombination product was introduced into 100 μL of E. coli Rosetta (DE3) competent cells; (2) ice bath for 10 minutes; (3) heat shock in a 42°C water bath for 1 minute and 30 seconds, and then quickly put it in ice and let it stand in an ice bath for 4 minutes; (4) 600 μL of non-resistant LB medium was added and mixed, and cultured at 37°C, 200 rpm for 1 hour; (5) centrifuged at 5000 rpm for 2 minutes to collect the bacteria; (6) the supernatant was removed, and the remaining 100-200 μL was mixed by pipetting and spread on a 0.05 mg / mL kanamycin LB resistance plate, and cultured at 37°C for about 12 hours. (7) Single clones were selected and placed in 0.05 mg / mL kanamycin resistance LB, and cultured at 200 rpm and 37°C for 12 hours, and then sent to the company for sequencing. The correct sequence was the positive transformant.

[0089] The engineered bacteria E. coli Rosetta (DE3)-pET28a (+)-12 × His_Δ106RnCHST3, E. coli Rosetta (DE3)-pET28a (+)-TrxA_Δ106RnCHST3, and E. coli Rosetta (DE3)-pET28a (+)-MBP_Δ106RnCHST3 constructed in Example 7 were selected and inoculated into 30 mL of fresh LB medium containing Kan and Cm resistance, and cultured at 37 ° C for 10 h. The inoculation volume was transferred to 150 mL of TB medium (containing Kan and Cm resistance) at 1% (v / v) and cultured at 37 ° C and 200 rpm until OD 600The induction temperature was 16℃ and the induction time was 18h. After induction, centrifuge at 4℃ and 8000rpm for 5min, wash the bacteria several times with PBS buffer, resuspend at 10mg / mL, ultrasonically break the bacteria for 10min (power 100W, ultrasonic 2s, intermittent 1.5s), high-speed refrigerated centrifugation at 4℃, 12000rpm, 10min. Take out the supernatant to sample the supernatant. Use a 1mL pipette tip to absorb 1mL PBS and resuspend, 4℃, 12000rpm, 2min (repeat twice), and take samples (precipitation). Add 10μL 4×SDS loading to the supernatant precipitate, boil it in a pot for 10min, and run protein gel. Protein gel identification revealed that the soluble expression level of E. coli Rosetta (DE3)-pET28a (+)-MBP_Δ106RnCHST3 was significantly increased. The expression of the three soluble tag fusion proteins in the cell was as follows: Figure 5 shown.

[0090] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A truncation of chondroitin 6-O-sulfotransferase, characterized in that Based on the amino acid sequence of chondroitin 6-O-sulfotransferase shown in SEQ ID NO.1, the amino acids at positions 1 to 11, 1 to 14, 1 to 58, 1 to 62, 1 to 65, 1 to 73, 1 to 106 or 1 to 131 of the N-terminus are truncated.

2. A truncation of chondroitin 6-O-sulfotransferase, characterized in that It has an amino acid sequence shown in any one of SEQ ID NOs. 2 to 9.

3. The chondroitin 6-O-sulfotransferase truncation according to claim 2, characterized in that: A solubility tag is also connected to the N-terminus of any one of the amino acid sequences shown in SEQ ID NO. 2 to 9; the solubility tag includes but is not limited to 12×His, TrxA or MBP.

4. A gene encoding the truncation of chondroitin 6-O-sulfotransferase according to any one of claims 1 to 3.

5. A recombinant vector carrying the gene according to claim 4.

6. A microbial cell expressing the chondroitin 6-O-sulfotransferase truncation according to any one of claims 1 to 3, or carrying the gene according to claim 4, or containing the recombinant vector according to claim 5.

7. A recombinant Escherichia coli, characterized in that The chondroitin 6-O-sulfotransferase truncation according to any one of claims 1 is expressed.

8. A method for preparing chondroitin sulfate C, characterized in that: The chondroitin 6-O-sulfotransferase truncation according to any one of claims 1 to 3, or the microbial cell according to claim 6, or the recombinant Escherichia coli according to claim 7 is subjected to a catalytic reaction in a reaction system containing chondroitin and 3'-phosphoadenosine-5'-phosphosulfate.

9. A method for increasing the expression of mouse chondroitin 6-O-sulfotransferase, characterized in that: The method comprises (a), (b) or (c): (a) truncating the amino acid sequence of the chondroitin 6-O-sulfotransferase; the truncation is to truncate the amino acids at positions 1 to 11, 1 to 14, 1 to 58, 1 to 62, 1 to 65, 1 to 73, 1 to 106 or 1 to 131 of the N-terminus of the chondroitin 6-O-sulfotransferase; (b) connecting a solubility-promoting tag 12×Hi, TrxA or MBP to the N-terminus of the chondroitin 6-O-sulfotransferase; (c) truncating the amino acid sequence of the chondroitin 6-O-sulfotransferase; the truncation is to truncate the amino acids 1 to 11, 1 to 14, 1 to 58, 1 to 62, 1 to 65, 1 to 73, 1 to 106 or 1 to 131 at the N-terminus of the chondroitin 6-O-sulfotransferase, and connecting a solubilizing tag 12×Hi, TrxA or MBP to the N-terminus of the truncated chondroitin 6-O-sulfotransferase.

10. Use of the chondroitin 6-O-sulfotransferase truncation according to any one of claims 1 to 3, or the recombinant microbial cell according to claim 6, or the recombinant Escherichia coli according to claim 7, or the method according to claim 8 in the preparation of a product containing chondroitin sulfate C.

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