CN116814578A - PpUGT5 recombinant protein, expression gene, expression cassette, recombinant vector, host bacterium and application - Google Patents

Abstract

The invention discloses a PpUGT5 recombinant protein, an expression gene, an expression cassette, a recombinant vector, a host bacterium and application of the PpUGT5 recombinant protein. The PpUGT5 recombinant protein comprises a polypeptide consisting of an amino acid sequence shown in SEQ ID No. 1. By combining with a specific embodiment, the PpUGT5 recombinant protein has the capability of catalyzing 6 'glycosylation of the trillion glycoside, realizes the glycosylation reaction of the trillion glycoside, generates the trillion glycoside 6' -O-glucoside, lays a foundation for the glycosylation research of the steroid saponin, and provides theoretical support for the formation research of the steroid saponin activity mechanism. The invention clones and verifies the glycosyltransferase function of the PpUGT5 recombinant protein for the first time, and the PpUGT5 recombinant protein can catalyze and synthesize the trilinear glycoside 6' -O-glucoside in vitro.

Description

PpUGT5 recombinant protein, expression gene, expression cassette, recombinant vector, host bacterium and application

Technical Field

The invention relates to the technical field of molecular biology, in particular to a PpUGT5 recombinant protein, an expression gene, an expression cassette, a recombinant vector, a host bacterium and application.

Background

Steroid saponin (steroidal saponins) is an important active ingredient of traditional Chinese medicine, is used for producing a plurality of steroid hormones and medicines, and widely exists in medicinal plants such as dioscorea nipponica, paris polyphylla, rhizoma anemarrhenae, lily, rhizoma polygonati, chinese yam and the like. The steroid saponin has remarkable pharmacological activity, such as anti-inflammatory, antibacterial, anti-tumor, antioxidant, antidepressant, liver protecting, etc. [1-3]. The steroid saponin is mainly formed by condensing steroid sapogenin and different glycosyl groups, the types of the linked glycosyl groups are various, and the steroid saponin mainly comprises glucose, rhamnose, galactose, arabinose and the like, and the difference of the number of the linked glycosyl groups and the linking mode is one of main reasons for generating different activities of the steroid saponin [4].

Therefore, researchers at home and abroad pay more attention to the analysis of molecular mechanisms of steroid saponin glycosylation, biosynthesis pathways thereof and the like, and a great deal of progress is made in the aspects of identification, functional analysis, modification and the like of steroid saponin glycosyltransferase at present [5-10]. Provides reference and theoretical support for the subsequent research of steroid saponins.

Glycosyltransferases are a vast family of supergenes in organisms, with similar domains, but no obvious homology. Glycosyltransferases are typically GT-B folds, comprising 2 distinct Rossmann domains-the C-terminal domain and the N-terminal domain-forming a beta/alpha/beta type structure [11]. The C-terminal domain is mainly responsible for identifying the sugar donor, and has high conservation degree. In general, plant glycosyltransferases contain a conserved domain of about 44 amino acids at the C-terminus, known as the plant secondary metabolite glycosyltransferase (PSPG) motif [12]. Among them, the 23 rd asparagine (N) and 44 th glutamine (Q) of amino acid determine the choice of glycosyltransferase protein to glucose donor to a great extent, the change of these two key sites will influence the catalytic activity of glycosyltransferase [13,14], this provides new thinking for glycosyltransferase to glycosyl donor selectivity, provide important reference for steroid saponin glycosylation reaction, lay foundation for obtaining more steroid saponin compounds with better biological activity.

Reference to the literature

[1] Ma Baiping, zhou Wen, feng Bing, etc. the research and development of Chinese medicinal saponins and bioconversion [ C ]. Fourth national microbiological resource academy and national microbiological resource platform running service meeting, discussion of the national microbiological resource platform, 2012:27-28.

[2]Upadhyay S,Jeena GS,Shikha,Shukla RK.Recent advances in steroidal saponins biosynthesis and in vitro production[J].Planta.2018,248(3):519-544.

[3] Luo Linming, agaric Pei Gang, huang Shaoguo, zhou Xiaojiang. Steroid saponin component of Lilium and its pharmacological activity research progress [ J ]. J.Chinese J.2018, 43 (7): 1416-1426.

[4] Zhang Xue, wang Xifu, zhao Ronghua, jie, gu, fuxing, cao Guanhua, he Sen. Pharmaceutical plant steroid saponin biosynthesis pathway research progress [ J ]. J.Chinese experimental prescription journal of laboratory, 2020,26 (14): 225-234.

[5]Huang W,He Y,Jiang R,Deng Z,Long F.Functional and structural dissection of a plant steroid 3-O-glycosyltransferase facilitated the engineering enhancement of sugar donor promiscuity[J].ACS Catal.2022,12,2927-2937.

[6]Song W,Zhang C,Wu J,Qi J,Hua X,Kang L,Yuan Q,Yuan J,Xue Z.Characterization of three Paris polyphylla glycosyltransferases from different UGT families for steroid functionalization[J].ACS Synth Biol.2022,11(4):1669-1680.

[7]Gao J,Xu Y,Hua C,Li C,Zhang Y.Molecular Cloning and Functional characterization of a sterol 3-O-glucosyltransferase involved in biosynthesis of steroidal saponins in Trigonella foenum-graecum[J].Front Plant Sci.2021,12:809579.

[8]Ye,T,Song W,Zhang JJ,An mei,Fen S,Yan S,Li J.Identification and functional characterization of DzS3GT,a cytoplasmic glycosyltransferase catalyzing biosynthesis of diosgenin 3-O-glucoside in Dioscorea zingiberensis[J].Plant Cell Tiss Organ Cult.2017,129:399-410.

[10]Hoang,N.H.,Huong,N.L.,Kim,B.et al.Kinetic studies on recombinant UDP-glucose:sterol 3-O-β-glycosyltransferase from Micromonospora rhodorangea and its bioconversion potential[J].AMB Expr.2016,6:52.

[11]Liang DM,Liu JH,Wu H,et al.Glycosyltransferases:mechanisms and applications in natural product development[J].Chem Soc Rev.2015,44(22):8350-8374.

[12]Rahimi S,Kim J,Mijakovic I,Jung KH,Choi G,Kim SC,Kim YJ.Triterpenoid-biosynthetic UDP-glycosyltransferases from plants[J].Biotechnol Adv.2019,37(7):107394.

[13]Shibuya M,Nishimura K,Yasuyama N,Ebizuka Y.Identification and characterization of glycosyltransferases involved in the biosynthesis of soyasaponinⅠin Glycine max.FEBS Lett.2010,584(11):2258-2264.

[14]Takagi K,Yano R,Tochigi S,Fujisawa Y,Tsuchinaga H,Takahashi Y,Takada Y,Kaga A,Anai T,Tsukamoto C,Seki H,Muranaka T,Ishimoto M.Genetic and functional characterization of Sg-4glycosyltransferase involved in the formation of sugar chain structure at the C-3position of soybean saponins.Phytochemistry.2018,156:96-105.

Disclosure of Invention

Based on this, it is necessary to provide a ppagt 5 recombinant protein having a glycosyltransferase function, which can be used for steroid saponin synthesis.

In addition, it is also necessary to provide the expression genes, expression cassettes, recombinant vectors, host bacteria and applications of the PpUGT5 recombinant proteins.

A ppagt 5 recombinant protein comprising a polypeptide consisting of the amino acid sequence shown in SEQ ID No. 1.

An expressed gene comprising a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID No.1, or a complementary strand of said polynucleotide.

In one embodiment, the polynucleotide has a sequence as set forth in SEQ ID No. 2.

An expression cassette comprising the above-described expression gene.

A recombinant vector comprising the above-described expressed gene.

In one embodiment, the recombinant vector is a cloning vector, which is pEASY-Blunt Simple Cloning Vector.

In one embodiment, the recombinant vector is an expression vector, which is a pGEX-4T-1 vector.

A host bacterium comprising the recombinant vector described above.

In one embodiment, the host bacterium is E.coli DH 5. Alpha. Or E.coli Rosetta-gami (DE 3) pLysS.

The PpUGT5 recombinant protein, the expression gene, the expression cassette, the recombinant vector and the application of the host bacteria in the field of steroid saponin compound synthesis.

By combining with a specific embodiment, the PpUGT5 recombinant protein has catalytic capability on the trillion glycoside, realizes glycosylation reaction of the trillion glycoside, generates trillion glycoside 6' -O-glucoside, lays a foundation for the glycosylation research of steroid saponin, and provides theoretical support for the formation research of the steroid saponin activity mechanism.

The invention clones and verifies the glycosyltransferase function of the PpUGT5 recombinant protein for the first time, and the PpUGT5 recombinant protein can catalyze and synthesize the trilinear glycoside 6' -O-glucoside in vitro.

In addition, the invention also provides an expression gene, an expression cassette, a recombinant vector and host bacteria of the PpUGT5 recombinant protein, and points out the application of the PpUGT5 recombinant protein in the field of synthesis of steroid saponins, lays a foundation for production by a bioengineering method, and provides a theoretical basis for glycosylation research of steroid saponins.

The PpUGT5 recombinant protein can be directionally modified in the future, and theoretical support is provided for the active formation mechanism of steroid saponin and the exploration of new compound synthesis.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Wherein:

FIG. 1 is an agarose gel electrophoresis diagram of PCR amplification products of a gene encoding a recombinant protein PpUGT 5; lane M is the amplified product (1389 bp) of the DNA molecular weight standard DL 2000,1, which is the gene encoding the recombinant protein PpUGT5.

FIG. 2 shows pGEX-4T-1 vector and multiple cloning sites, highlighting the cleavage sites for ligation of recombinant plasmids in this scheme.

FIG. 3 is a SDS-PAGE electrophoresis containing a recombinant PpUGT5 protein; lane M is Page-roller pre-dye protein Ladder,1 is induced pGEX-4T-1-Rosetta-gami (DE 3) pLysS protein, 2 is uninduced pGEX-PpUGT5-Rosetta-gami (DE 3) pLysS protein, 3 is pGEX-PpUGT5-Rosetta-gami (DE 3) pLysS cell supernatant protein after IPTG induction, 4 is pGEX-PpUGT5-Rosetta-gami (DE 3) pLysS cell precipitate after IPTG induction, 5 is purified GST-tagged PpUGT5 protein with molecular weight 78.49kDa.

FIG. 4 is a liquid chromatographic analysis of a substrate of trillion after the reaction of trillion by PpUGT5 recombinant protein, UDP-glucose as sugar donor, acquisition time (min) on the abscissa and electrical signal (mAU) on the ordinate.

FIG. 5a is a mass spectrum analysis of the product of the PpUGT5 recombinant protein catalyzed by the reaction of trillin, UDP-glucose as the sugar donor, the abscissa as mass-to-charge ratio and the ordinate as ionic strength.

FIG. 5b is a liquid chromatography analysis chart of the product of the PpUGT5 recombinant protein catalyzed delay grass glycoside reaction, UDP-glucose is a sugar donor, the abscissa is the acquisition time (min), and the ordinate is the electrical signal (mAU).

FIG. 6 shows the results of the hydrogen and carbon spectra of the glycosylation products.

FIG. 7 is a flow chart of PpUGT5 catalyzing glycosylation of trillin 6' -O-glucoside.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the implementations described are only some, but not all embodiments of the invention. All other embodiments, based on the embodiments of the invention, which would be apparent to one of ordinary skill in the art without having to do pwz with the inventive effort, are within the scope of the invention.

The invention discloses a PpUGT5 recombinant protein of an embodiment, which comprises a polypeptide consisting of an amino acid sequence shown in SEQ ID No. 1.

By combining with a specific embodiment, the PpUGT5 recombinant protein has catalytic capability on the trillion glycoside, realizes glycosylation reaction of the trillion glycoside, generates the trillion glycoside 6' -O-glucoside, lays a foundation for the glycosylation research of the steroid saponin, and provides theoretical support for the formation research of the steroid saponin activity mechanism.

The invention clones and verifies the glycosyltransferase function of the PpUGT5 recombinant protein for the first time, and the PpUGT5 recombinant protein can catalyze and synthesize the trilinear glycoside 6' -O-glucoside in vitro.

The PpUGT5 recombinant protein can be directionally modified in the future, and theoretical support is provided for the active formation mechanism of steroid saponin and the exploration of new compound synthesis.

In this embodiment, the recombinant protein PpUGT5 may be referred to as a recombinant protein PpUGT5, which is a recombinant protein of trillin 6' -O-glucosyltransferase.

The invention also discloses an expression gene of an embodiment, which comprises a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID No.1, or a complementary strand of the polynucleotide.

Specifically, in this embodiment, the sequence of the polynucleotide is shown in SEQ ID No. 2.

Specifically, the expressed gene is separated from rhizome of paris polyphylla plant, and the PpUGT5 recombinant protein obtained by expressing the expressed gene is uridine diphosphate glucosyltransferase.

The full length of the ORF sequence of the expressed gene is 1398 nucleotides, and the encoded PpUGT5 recombinant protein contains 465 amino acids and contains a conserved domain of glycosyltransferase, namely PSPG-box domain.

In this embodiment, the whole expression gene may be referred to as the trillin glucosyltransferase PpUGT5 gene.

In the invention, the preparation method of the PpUGT5 recombinant protein comprises the following steps: the expression gene was ligated to pGEX-4T-1 vector to construct recombinant plasmid pGEX-PpUGT5. And (3) using escherichia coli to induce pGEX-PpUGT5 recombinant protein expression to obtain the PpUGT5 recombinant protein.

The PpUGT5 recombinant protein can catalyze the glycosylation reaction of the trillion glycoside 6 'to synthesize the trillion glycoside 6' -O-glucoside.

The invention also discloses an expression cassette comprising the expression gene.

The invention also discloses a recombinant vector comprising the expressed gene.

In one embodiment, the recombinant vector is a cloning vector, which is pEASY-Blunt Simple Cloning Vector.

In another embodiment, the recombinant vector is an expression vector, and the expression vector is a pGEX-4T-1 vector.

The invention also discloses a host bacterium comprising the recombinant vector.

In one embodiment, the host bacterium may be a host bacterium suitable for cloning of a candidate gene, such as E.coli DH 5. Alpha.

In another embodiment, the host bacterium may also be a host bacterium for recombinant protein expression, such as E.coli Rosetta-gami (DE 3) pLysS.

The PpUGT5 recombinant protein, the expression gene, the expression cassette, the recombinant vector and the host bacteria disclosed by the invention can be applied to the field of steroid saponin compound synthesis.

The invention also provides an expression gene, an expression cassette, a recombinant vector and host bacteria of the PpUGT5 recombinant protein, and points out the application of the PpUGT5 recombinant protein in the field of synthesis of steroid saponins, lays a foundation for production by a bioengineering method, and provides a theoretical basis for glycosylation research of steroid saponins.

The following are specific examples.

EXAMPLE 1 cloning of the Gene encoding the recombinant protein PpUGT5

1. Experimental materials

Paris yunnanensis (Paris polyphylla SMITH var. Yunnanensis (Franch.) hand. Mazz.) is picked up in Yunnan Kunming plantations, and the plants are in good condition and grow consistently. Different tissues of paris polyphylla, including roots, stems, leaves and fruits, are subjected to liquid nitrogen quick freezing three times each group.

2. Vectors and strains

Coli (Escherichia coli) competent cells Trans 5a Chemically Competent Cell (CD 201) were purchased from beijing full gold biotechnology inc.

Coli Rosetta-gami (DE 3) pLysS competent cells (ZC 1212) were purchased from Beijing bang nationality biological gene technologies Inc.

The cloning vector pEASY-Blunt Simple Cloning Vector (CB 111) was purchased from Beijing full gold Biotechnology Co., ltd.

Prokaryotic expression vector pGEX-4T-1 was purchased from Beijing village allied biological gene technologies Co.

3. Solution preparation

(1) LB medium (500 mL): accurately weighing 5g of tryptone, 2.5g of yeast extract and 5g of sodium chloride, fixing the volume to 500mL by using ultrapure water, adding 7.5g of agar powder into a solid culture medium, sterilizing at 121 ℃ for 15min under high pressure, cooling to normal temperature, and storing in a refrigerator at 4 ℃ for later use. Amp solution (100 mg/mL): 2g of Amp powder was weighed accurately and dissolved in 20mL of sterilized water, sterilized with a 0.22 μm sterile filter, and stored in a refrigerator at-20℃until use (working concentration: 100. Mu.g/mL).

(2) Isopropyl thio- β -D-galactoside (IPTG) (500 mM): weighing 5.95g of IPTG, dissolving in sterilized ultrapure water, fixing the volume to 50mL, filtering for sterilization, sub-packaging into small tubes, and preserving at-20 ℃.

(3) Tris-HCl buffer (pH 7.5, 100 mM): 1.1214g of Tris is weighed, added with water to 90mL, stirred and dissolved uniformly, added with HCl to adjust the pH to 7.5, and added with water to fix the volume to 100mL.

Cloning of the Gene encoding the recombinant protein PpUGT5

Based on the analysis of Yunnan paris polyphylla transcriptome data, the PpUGT5 recombinant protein coding gene expressed in rhizome is found, and gene cloning and in vitro protein expression are carried out in order to determine the function of the PpUGT5 recombinant protein coding gene in the paris polyphylla saponin biosynthesis pathway.

(1) Extraction of Total RNA

The RNA of the paris polyphylla leaves is extracted by using a QIAGEN RNeasy Plant Mini Kit kit (74903) operation instruction, and the extraction process should be kept on ice all the time to operate, so that RNA degradation is avoided.

(2) Reverse transcription reaction

Full-length cDNA of Siraitia grosvenorii was synthesized using the reverse transcription kit TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (AU 311) from Beijing full-length gold Biotechnology Co., ltd, and the specific procedures are shown in Table 1.

TABLE 1 reaction System for first Strand Synthesis of cDNA

Lightly blowing and mixing the reaction solution by a micropipette, and stirring at 50 ℃ for 15min;85 ℃,5s.

(3) PCR amplification of PpUGT5 recombinant protein coding gene

The CE Design software is used for respectively designing full-length amplification primers of the PpUGT5 recombinant protein coding gene, and the primer sequences are SEQ: NO.3 and SEQ: NO.4. Candidate sequences were amplified by PCR using cDNA as a template, and the PCR system is shown in Table 2.

TABLE 2PCR reaction System

PCR reaction temperature procedure:

the gel electrophoresis was performed on 5. Mu.L of the PCR product, and the detection result is shown in FIG. 1.

Referring to FIG. 1, it can be seen that the length of the encoding gene of PpUGT5 is 1398bp.

(4) PCR product purification

The PCR product was subjected to DNA fragment purification, and the procedure was performed according to the instructions of the DNA fragment purification kit (EasyPure PCR Purification Kit, EP 101) of Beijing full gold Biotechnology Co., ltd.

(5) Cloning vector ligation reactions

The purified PCR product was subjected to cloning vector ligation, and the specific reaction system is shown in Table 3.

TABLE 3 reaction System for cloning genes

The reaction solutions in the table above were mixed thoroughly and reacted at room temperature for 5min, and E.coli competent Trans 5. Alpha. Chemically Competent Cell was directly transformed.

(6) Screening of transformed E.coli Trans5α Chemically Competent Cell and Positive clones

The cloning vector containing the target gene is transformed into Trans5 alpha Chemically Competent Cell, after culturing for 12 hours at 37 ℃, monoclonal is selected and added into LB liquid culture medium with kana resistance, after culturing for 6-8 hours at 37 ℃ and 180r/min, positive clones containing the target fragment are identified and screened by PCR, and the sequencing identification of bacterial liquid with the electrophoresis result meeting the expectancy is completed by Shanghai biological technology company.

After sequencing, the sequence of the PpUGT5 recombinant protein coding gene is shown as SEQ ID No.2, and the sequence of the PpUGT5 recombinant protein coded by the PpUGT5 recombinant protein coding gene is shown as SEQ ID No. 1.

(7) Extraction of cloning vector plasmid DNA

The bacterial liquid with correct sequencing result is added into LB liquid culture medium (Amp resistance), shake culture is carried out for 12 hours at 37 ℃, bacteria are collected by centrifugation, and recombinant plasmids are extracted, and the operations are carried out with specific reference to the specification of a Tiangen ultrapure plasmid extraction kit (TIANpure Mini Plasmid Kit, DP 104).

EXAMPLE 2 construction of expression vector for encoding Gene of PpUGT5 recombinant protein

(1) Expression vector construction primer design

Referring to FIG. 2, a specific primer with homologous sequences near the double cleavage sites of the prokaryotic expression vector pGEX-4T-1 is designed, and the primer sequence is shown as SEQ: no.5 and SEQ: NO. 6.

(2) PCR amplification and purification

The correctly sequenced cloning plasmid was used as template, with SEQ: no.5 and SEQ: no.6 was subjected to PCR amplification, and the specific procedure was as shown in (3) Table 2 and the PCR reaction temperature program in the cloning of the 4.PpUGT5 recombinant protein-encoding gene in example 1, and the obtained PCR product was subjected to the procedure using the DNA fragment purification kit instruction of Beijing full-scale gold biotechnology Co., ltd.

(3) Double cleavage reaction of pGEX-4T-1 vector plasmid

The pGEX-4T-1 vector plasmid was digested with XmaI and SalI restriction enzymes to obtain linearized vectors, the specific procedures of which are shown in Table 4.

Table 4 double cleavage reaction System

The reaction was incubated at 37℃for 2.5h.

(4) Enzyme-cut product gum recovery

The linearized vector was purified using Beijing full gold rapid gel recovery kit (EasyPure Quick Gel Extraction Kit, EG 101) and operated according to the instructions.

(5) Seamless cloning recombination reactions

pGEX-PpUGT5 recombinant vector was constructed by using NEBuilder HiFi DNA Assembly Master Mix kit (NEB#e2621 x), and the specific reaction system was as shown in Table 5.

TABLE 5 ligation reaction System

Mix gently and incubate at 50℃for 15min. Cooling to 4 ℃ or immediately cooling on ice.

(6) Transformation of recombinant vectors

The 5 mu L reaction solution is transformed into escherichia coli competent cells in Trans5 alpha Chemically Competent Cell by a freeze thawing method, the specific operation is shown as cloning (6) of a 4.PpUGT5 recombinant protein coding gene in example 1, and bacterial liquid sequencing identification is completed by Shanghai Biotechnology company.

(7) Extraction of recombinant plasmid DNA

The bacterial liquid with correct sequencing result is added into LB liquid culture medium (Amp resistance), shake-cultured for 12h at 37 ℃, and then the bacterial liquid is centrifugally collected to extract recombinant plasmid pGEX-PpUGT5, and the plasmid extraction method is operated according to the specification (Tiangen ultra-pure plasmid extraction kit).

EXAMPLE 3 prokaryotic expression of the PpUGT5 recombinant protein

(1) Recombinant plasmid transformation expression vector Rosetta-gami (DE 3) pLysS

The pGEX-PpUGT5 recombinant plasmid and pGEX-4T-1 are transformed into competent cells of escherichia coli Rosetta-gami (DE 3) pLysS by a freeze thawing method, positive cloning is verified by colony PCR, and positive cloning bacterial liquid 1:1 adding 50% glycerol, and storing at-80deg.C.

(2) Protein-induced expression

Adding 50 mu L of bacterial liquid into 10mL of LB culture medium (Amp), and shake culturing at 37 ℃ for overnight; adding 2mL of the overnight cultured bacterial liquid into 300mL of LB culture medium (Amp), shake culturing at 37 ℃ until OD600 = 0.6, adding IPTG with a final concentration of 1mM, performing induction culture at 17 ℃ for 24 hours (overnight), centrifugally collecting bacterial cells at 4 ℃, adding 20mL of 4 ℃ precooled PBS phosphate buffer (PH 6.8, 1M) to suspend bacterial cells, ultrasonically crushing for 20 minutes, stopping 2 seconds after working for 6 seconds, ultrasonically treating for 15 minutes until the bacterial liquid becomes transparent, centrifuging at 12000rpm for 30 minutes, and collecting supernatant to obtain crude enzyme protein.

(3) Protein purification

Glutathione Beads (Hengzhou Tiandi and Biotechnology Co., ltd.; cat. No. SA 008010) was packed into a chromatography column, column equilibrated with 5 column volumes of equilibration solution (40mM NaCl,2.7mM KCl,10mM Na2HPO4,1.8mM KH2PO4,pH 7.4,1mM DDT, total volume 1L), incubated with the supernatant for 2h, the effluent was collected, eluted with an eluent (50 mM Tris-HCl,150mM NaCl,10mM reduced glutathione, 1mM DDT, pH 8.0, total volume 1L), the effluent was collected by hands in fractions of 2mL each tube, and protein expression and purification were detected by SDS-PAGE, and the results are shown in FIG. 3.

Referring to FIG. 3, it can be seen that pGEX-PpUGT5 recombinant plasmid is transformed into expression strain Rosetta-gami (DE 3) pLysS, and is expressed after IPTG induction, and purified to obtain purer recombinant protein, and the recombinant protein band size is consistent with the predicted, and after adding a recombinant tag, there is an obvious recombinant protein band at about 78.49kDa. The purified protein can be used for further enzymatic analysis.

The band-specific, high concentration of the tube protein was concentrated by ultrafiltration tube and the purified protein was preserved with 10% (v/v) glycerol (-80 ℃).

EXAMPLE 4 functional analysis of PpUGT5 recombinant protein

(1) In vitro enzymatic reaction

1mM of trillin, 5mM of UDP-Glucose (UDP-Glc), 50mM of Tris (pH 8.0) and 1mM of MgCl are added to the reaction mixture ₂ The purified protein was used to make up to 100. Mu.L, reacted at 37℃for 12 hours, then the enzyme activity reaction was terminated by adding an equal volume of methanol, the reaction solution was spin-dried, and then dissolved in 100. Mu.L of chromatographic methanol, and centrifuged at 12,000rpm for 10 minutes, and the supernatant was used for the subsequent measurement.

(2) HPLC-ESI-QTOF MS/MS assay

The product and substrate were detected in this experiment using Agilent 1290 Infinicity II and Agilent 6546LC/Q-TOF, resulting in FIGS. 4, 5a and 5b. The specific measurement parameter conditions are shown in Table 6.

TABLE 6HPLC-QQQ-MS/MS determination parameter Table

(3) Nuclear magnetic resonance spectroscopy (Nuclear magnetic resonance spectroscopy, NMR)

The obtained new product was enriched and analyzed by nuclear magnetic resonance spectroscopy to obtain the new product of PpUGT5 recombinant protein catalytic production of trillionin, and the structure was identified to obtain FIG. 6.

According to the analysis of the results of FIG. 4, FIG. 5a and FIG. 5b, pGEX-4T-1 empty load was found as a control, and the PpUGT5 recombinant protein was able to catalyze the production of a new product of trillin with UDP-glucose as the sugar donor, the acquisition time was 5.912min, the charge-to-mass ratio was 739.4284 (M+H) ⁺ ) And the analysis based on MS/MS results found the chemicalThe compound was cleaved by one RDA followed by cleavage of two glucoses, so we speculate that the compound might be trillin 6' -O-glucoside.

The structure of the glycosylated product was identified by nuclear magnetic resonance spectroscopy (Nuclear magnetic resonance spectroscopy, NMR) analysis after enrichment and purification, and the glycosylated product was trilineoside-6' -glucoside by combining hydrogen spectrum and carbon spectrum analysis (figure 6). The results of the nuclear magnetic analysis are as follows: 1H NMR (500 MHz, CD3 OD): δH 5.39 (1H, d, J=5.2, H-6), 4.39 (1H, overlay, H-16), 4.39 (1H, d, J=7.8 Hz, H-1 '), 4.38 (1H, d, J=7.8 Hz, H-1 "), 4.12 (1H, dd, J=11.6, 2.0Hz, H-6' a), 3.86 (1H, dd, J=11.9, 1.9Hz, H-6" a), 3.78 (1H, dd, J=11.6, 5.6Hz, H-6' b), 3.66 (1H, dd, J=11.9, 5.3Hz, H-6 "b), 3.55 (H, overlay, H-5 '), 3.46 (1H, m, H-3), 3.45 (1H, m, H-26 a), 3.43 (2H, 2H-4 ', 3.4H-4 '. 3.34 (2H, overlay, H-2', H-3"), 3.33 (1H, overlay, H-26b "), 3.28 (1H, overlay, H-4"), 3.27 (1H, m, H-5 "), 3.20 (1H, m, H-2") 2.43 (1H, m, H-4 a), 2.33 (1H, m, H-4 b), 2.0 (2H, m, H-8), 1.92 (2H, m, H-2), 1.91 (2H, m, H-20), 1.87 (2H, m, H-1), 1.75 (1H, m, H-17), 1.05 (3H, s, H-19), 0.97 (3H, d, j=7.0, H-21), 0.82 (3H, s, H-18), 0.79 (3H, d, j=6.27-4 hz). 13C (125 MHz, CD3 OD): delta C142.0 (C-5 '), 122.6 (C-6'), 110.6 (C-22 '), 104.7 (C-1'), 102.2 (C-16 '), 80.1 (C-3'), 78.0 (C-3 '), 77.9 (C-5'), 77.0 (C-5 '), 75.1 (C-2'), 71.6 (C-4 '), 71.5 (C-4'), 69.1 (C-6 '), 67.9 (C-26), 63.8 (C-17'), 62.8 (C-6 '), 57.8 (C-14), 51.6 (C-9), 42.9 (C-20), 41.4 (C-13), 40.9 (C-12), 39.8 (C-4), 38.0 (C-1), 38.0 (C-2'), 75.1 (C-32), 32 (C-8), 32 (C-32), 32.8 (C-32), 31.9 (C-32), 31.8 (C-32), 32 (C-4), and (C-32) (32.8 (C-32). 14.9 (C-21).

In combination with the above analysis, the PpUGT5 recombinant protein can catalyze the glycosylation reaction of the trillion glycoside 6 'to generate the trillion glycoside 6' -O-glucoside, and the specific catalysis flow is shown in FIG. 7.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. A recombinant ppagt 5 protein comprising a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1.

2. An expressed gene comprising a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID No.1, or a complementary strand of said polynucleotide.

3. The expressed gene of claim 2, wherein the polynucleotide has a sequence as shown in SEQ ID No. 2.

4. An expression cassette comprising the expressed gene of claim 2 or 3.

5. A recombinant vector comprising the expressed gene of claim 2 or 3.

6. The recombinant vector according to claim 5, wherein the recombinant vector is a cloning vector, and the cloning vector is pEASY-Blunt Simple Cloning Vector.

7. The recombinant vector according to claim 5, wherein the recombinant vector is an expression vector, and the expression vector is a pGEX-4T-1 vector.

8. A host bacterium comprising the recombinant vector according to any one of claims 5 to 7.

9. The host bacterium according to claim 8, wherein the host bacterium is E.coli DH 5. Alpha. Or E.coli Rosetta-gami (DE 3) pLysS.

10. Use of the ppagt 5 recombinant protein according to claim 1, the expression gene according to claim 2 or 3, the expression cassette according to claim 1, the recombinant vector according to claim 5, 6 or 7 and the host bacterium according to claim 8 or 9 in the field of steroid saponin compound synthesis.