Efficient expression and purification method of 5 '-3' exonuclease Xrn1

Abstract

The invention discloses a high-efficiency expression and purification method of 5 '-3' exonuclease, which comprises the following steps: based on a Bac-to-Bac system, adding a tag and a TEV restriction enzyme cutting site to optimized 5 '-3' exonuclease Xrn1, then connecting the 5 '-3' exonuclease Xrn1 to a multiple cloning site behind a Polyhedrin promoter of a pFastBac expression vector to prepare expression baculovirus plasmids, and carrying out purification on the expression baculovirus plasmids to obtain the 5 '-3' exonuclease. Then transfecting the recombinant baculovirus particles into insect cells to generate recombinant baculovirus particles; finally, the 5 '-3' exonuclease Xrn1 is obtained through nickel column affinity chromatography, Heparin affinity chromatography and gel filtration chromatography purification, the invention provides a more economical and practical technical route for synthesizing the 5 '-3' exonuclease Xrn1, and a technical foundation is laid for large-scale production of the 5 '-3' exonuclease.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an efficient expression and purification method of 5'-3' exoribonuclease Xrn1. Background Art

[0002] 5'-3' exoribonucleases (Xrn1) are a large family of conserved enzymes associated with important functions such as RNA metabolism and RNA interference during gene expression. They are present in all eukaryotes and exhibit different independent functions at different intracellular processing levels, including RNA degradation, mRNA transport, rRNA and small nucleolar RNA processing, etc. Xrn1 can continuously degrade RNA with 5'-terminal monophosphate, but cannot act on RNA with 5'-terminal triphosphate, 5' cap structure, or 5' hydroxyl group. The 5' end of all eukaryotic mRNAs is capped, which is crucial for mRNA stability, splicing, polyadenylation, nuclear export, and translation efficiency. The removal of the cap structure is a highly regulated process. The decapping enzyme cleaves 7-methyl-guanosine diphosphate from the mRNA to generate 5'-monophosphate mRNA. The 5'-monophosphate structure is then specifically recognized and cleaved by the 5'-3' exoribonuclease Xrn1, triggering RNA degradation and decapping.

[0003] 5'-3' exoribonuclease (Xrn1) can specifically hydrolyze 5'-terminal monophosphate RNA and has been widely used in gene transcriptome research, RNAi mechanism research and sRNA cloning. As a new tool enzyme in molecular biology, the emergence of highly active and widely used recombinant enzymes will greatly facilitate related research. Therefore, it is of great significance to screen an expression system suitable for efficient expression of active Xrn1 recombinant protein, optimize the expression conditions, separate and purify the recombinant protein, and identify its biological activity. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an efficient method for expressing and purifying 5'-3' exoribonuclease Xrn1.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. An efficient expression and purification method for 5'-3' exoribonuclease Xrn1, the method comprising the following steps: based on the Bac-to-Bac system, a TEV restriction site and an 8×His tag are connected to the C-terminus of the optimized Xrn1 gene sequence, and the fragment is connected to the multiple cloning site after the Polyhedrin promoter of the pFastBac expression vector, and recombined with the parent bacoid virus plasmid in the DH10Bac Escherichia coli competent cells to form an expression bacoid virus plasmid; then, the expression bacoid virus plasmid is transfected into insect cells to generate recombinant bacoid virus particles, the insect cells are crushed under high pressure and the supernatant is collected by centrifugation to obtain a crude extract; finally, the crude extract is purified by nickel column affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography to obtain the 5'-3' exoribonuclease Xrn1.

[0007] Preferably, in the present invention, the optimized Xrn1 gene sequence is shown as SEQ ID NO.1.

[0008] Preferably, the pFastBac expression vector is a pFastBac Dual vector.

[0009] Preferably, the insect cells are Sf9, Sf21 or High Five cells.

[0010] Preferably, during transfection, the density of insect cells is 3×10 6 / mL, and the amount of expression bacmid added was 0.15% (v / v).

[0011] Preferably, the eluent used in the nickel column affinity chromatography is: 300 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 250 mM imidazole pH 8.0.

[0012] Preferably, the buffer used in Heparin affinity chromatography is:

[0013] Low salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP;

[0014] High salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 1 M NaCl.

[0015] Preferably, the buffer used in the gel filtration chromatography is: 300 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 mM DTT.

[0016] The beneficial effects of the present invention are as follows: the present invention provides a method for efficiently expressing an active 5'-3' exoribonuclease Xrn1 using an insect baculovirus expression system, connecting a TEV restriction site and an 8×His tag to the C-terminus of an optimized Xrn1 gene sequence, and connecting the fragment to the multiple cloning site after the Polyhedrin promoter of the pFastBac Dual expression vector, and recombining with the parental baculovirus plasmid in the DH10Bac Escherichia coli competent cells to form an expression baculovirus plasmid; then, the expression baculovirus plasmid is transfected into insect cells to generate recombinant baculovirus particles; and finally, the 5'-3' exoribonuclease Xrn1 with uniform molecular weight and good properties is successfully purified by nickel column affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography. The 5'-3' exoribonuclease Xrn1 prepared by the present invention has similar enzymatic activity to commercial Xrn1, has the activity of degrading mRNA of 5'-terminal monophosphate, and is concentration-dependent. The invention provides a more economical and practical technical route for synthesizing 5'-3' exoribonuclease Xrn1, which lays a technical foundation for large-scale production of 5'-3' exoribonuclease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0018] Figure 1 The experimental flow chart is shown in Figure 2.

[0019] Figure 2 The expression and purification results of 5'-3' exoribonuclease Xrn1; a: gel filtration chromatogram of Xrn1 (1-1246); b: SDS-PAGE diagram.

[0020] Figure 3 Agarose Gel images and grayscale analysis statistics of mRNA degradation by 5'-3' exoribonuclease Xrn1 at different concentrations (Xrn1 is the recombinant protein prepared by the present invention, and NEB-Xrn1 is a commercial 5'-3' exoribonuclease). DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0022] The experimental flow chart of the present invention for in vitro expression and purification of 5'-3' exoribonuclease Xrn1 and identification of its activity is as follows: Figure 1 shown.

[0023] Example 1. Construction of expression vector

[0024] Amplify the optimized 5'-3' exoribonuclease Xrn1 (1-1246, SEQ ID NO.2) (UniProtID XRN1_YEAST, P22147) gene (SEQ ID NO.1), connect the TEV restriction site and 8×His tag to the C-terminus of the Xrn1 gene sequence, and ligate the fragment to pFastBac TM The recombinant plasmid pFastBacDual-pH-Xrn1(1-1246)-TEV was obtained by inserting the polyhedrin promoter into the multiple cloning site of the Dual expression vector. CS -His8.

[0025] Example 2. Preparation of recombinant baculovirus

[0026] With reference to the Bac-to-Bac baculovirus expression system operation manual, the recombinant plasmid prepared in Example 1 was transferred into DH10Bac E. coli competent cells, and recombined with the parental baculovirus plasmid in the bacteria to form an expression baculovirus plasmid for expressing the recombinant protein.

[0027] Example 3. Expression of recombinant protein

[0028] The density of Sf9 cells used for protein expression was 3×10 6 / ml, the amount of the expression bacmid prepared in Example 2 added was 0.15% (v / v), the temperature was 27°C, and the rotation speed was 120 rpm. Generally, after 60 hours of expression, the cell precipitate was collected by centrifugation at 4°C, 3000 rpm for 15 minutes.

[0029] Example 4. Purification of recombinant protein

[0030] 4.1 The cell pellet obtained in Example 3 was resuspended in Buffer A (300 mM NaCl, 50 mM Tris pH 8.0, 0.5 mM TCEP, 1 mM PMSF, 0.8 μM Aprotinin, 1 μM Pepstatin, 10 μM Leupeptin).

[0031] 4.2 Use a high pressure disruptor to disrupt the cells, 500 bar, 2 min, 4 °C.

[0032] 4.3 Centrifuge at 17000 rpm, 4°C for 40 minutes and collect the supernatant.

[0033] 4.4 Ni-NTA affinity chromatography

[0034] (1) Incubate the supernatant with Ni-NTA column at 4°C for 40 minutes.

[0035] (2) Wash with Wash buffer (300 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 20 mM Mimidazole pH 8.0) for 10 column volumes.

[0036] (3) Add appropriate amount of Elute buffer (300mM NaCl, 50mM Tris-HCl pH 8.0, 0.5mM TCEP, 250mM imidazole pH 8.0) in small amounts and multiple times.

[0037] 4.5 Heparin affinity chromatography (removal of impurities and TEV enzyme)

[0038] Low salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP.

[0039] High salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 1 M NaCl.

[0040] Collect the corresponding number of tubes of target protein and concentrate to 2 ml using a 100 KDa cut-off tube.

[0041] 4.6 Gel filtration chromatography (Superdex 200 10 / 300GL)

[0042] Buffer B: 300mM NaCl, 50mM Tris-HCl pH 7.5, 2mM DTT.

[0043] The expression and purification results of 5'-3' exoribonuclease Xrn1 are shown in Figure 2 As shown, each lane in Figure b corresponds to the sample with the elution peaks marked at positions 13-20 in Figure a. As can be seen from the figure, the 5'-3' exoribonuclease pFastBacDual-pH-Xrn1(1-1246)-TEV was constructed. CS -His8 recombinant plasmid was expressed in the eukaryotic insect cell Sf9, and finally successfully purified by nickel column affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography to obtain a 5'-3' exoribonuclease Xrn1 protein with uniform molecular weight and good properties, with a yield of up to 3 mg / L.

[0044] Example 5. Identification of enzyme activity of recombinant protein

[0045] 5.1 RNA 5′ pyrophosphohydrolase (RppH) processing of mRNA

[0046]

[0047] 5.2 Treatment of 5'-terminal monophosphate mRNA with Xrn1 recombinant protein prepared in Example 4

[0048]

[0049] The results of the enzymatic activity identification of 5'-3' exoribonuclease Xrn1 are as follows Figure 3 As shown in the figure, it can be seen that the activity of the Xrn1 expressed and purified by the present invention is similar to that of the commercial 5'-3' exoribonuclease Xrn1 (NEB-Xrn1) purchased from NEB, and has the activity of degrading mRNA with 5'-terminal monophosphate in a concentration-dependent manner.

[0050] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. An efficient expression and purification method for 5'-3' exoribonuclease Xrn1, characterized in that: The method comprises the following steps: based on the Bac-to-Bac system, a TEV restriction site and an 8×His tag are connected to the C-terminus of the optimized Xrn1 gene sequence, and the gene fragment is connected to the multi-cloning site behind the Polyhedrin promoter of the pFastBac expression vector, and recombined with the parent bacoid virus plasmid in the DH10Bac Escherichia coli competent cells to form an expression bacoid virus plasmid; then, the expression bacoid virus plasmid is transfected into insect cells to generate recombinant bacoid virus particles, the insect cells are crushed under high pressure and the supernatant is collected by centrifugation to obtain a crude extract; finally, the crude extract is purified by nickel column affinity chromatography, Heparin affinity chromatography, and gel filtration chromatography to obtain a 5'-3' exoribonuclease Xrn1 protein.

2. The method according to claim 1, characterized in that: The optimized Xrn1 gene sequence is shown as SEQ ID NO.

1.

3. The method according to claim 1, characterized in that: The pFastBac expression vector is a pFastBac Dual vector.

4. The method according to claim 1, characterized in that: The insect cells are Sf9, Sf21 or High Five cells.

5. The method according to claim 1, characterized in that: During transfection, the density of insect cells was 3 × 10 6 / mL, and the amount of expression bacmid added was 0.15% (v / v).

6. The method according to claim 1, characterized in that: The eluent used for nickel column affinity chromatography was: 300 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.5 mM TCEP, 250 mM imidazole pH 8.

0.

7. The method according to claim 1, characterized in that: The buffer used for Heparin affinity chromatography is: Low salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP; High salt buffer: 25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 1 M NaCl.

8. The method according to claim 1, characterized in that: The buffer used for gel filtration chromatography was: 300 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 mM DTT.

Images