CN113045630A - Method for purifying hepatitis B virus X protein - Google Patents

Abstract

The invention discloses a method for purifying hepatitis B virus X protein, which comprises the steps of carrying out fusion expression on hepatitis B virus X protein to be researched, CL7 protein and His label, obviously improving the soluble expression amount of target protein by utilizing the solubility-promoting characteristic and optimized expression condition of CL7 protein, removing the CL7 protein subjected to fusion expression by utilizing 3C protease enzyme digestion and carrying out affinity chromatography purification twice, realizing the purification of the hepatitis B virus X protein, and finally obtaining the X protein with higher purity and without the label. By adopting the purification method of the hepatitis B virus X protein, the fusion of the CL7 label can obviously improve the soluble expression quantity of the hepatitis B virus X protein, and avoid the troubles of the post-treatment renaturation of the inclusion body protein and the instability of protease and inhibitor thereof to the activity thereof.

Description

Method for purifying hepatitis B virus X protein

Technical Field

The invention relates to the technical field of protein purification, in particular to a method for purifying hepatitis B virus X protein.

Background

Hepatitis B is a disease caused by infection with Hepatitis B Virus (HBV), mainly characterized by hepatoinflammatory disease, and may cause multiple organ damage. At present, about 1 hundred million people in China are hepatitis B virus carriers, which account for about 8% -10% of the total population in China, and about 2000 million chronic hepatitis B patients are still one of the most social-burdened diseases in China until today.

HBV is a lysogenic virus of a hepadnavirus family, the genome is in a relaxed circular double-chain structure, the genome can encode a plurality of proteins with different biological functions, and contains four Open Reading Frames (ORFs), wherein a product (HBx) protein encoded by an X region is a multifunctional regulatory protein, has a wide trans-activation function, and has definite effects on intracellular signal transduction, virus replication and transcription, cell cycle progression, cell proliferation and apoptosis, protein degradation, stable inheritance of stem cells and the like.

The X gene is the smallest one of 4 open reading frame frameworks of the HBV genome, is positioned between 1374-1838 nucleotides, has the length of 452-465 bp, is the most obvious overlapped region in the structure and the function of the HBV genome, can code at least 3 proteins with different sizes, has various transcription regulation activities, but has different functions in regulation. The transactivation of HBx is closely related to the conservation and secondary structure of its amino acid sequence, HBx is composed of 145-154 amino acids, about 52% of which are hydrophobic amino acids, 9 cysteine residues play a very important role in the structure of HBx protein, each cysteine residue forms a disulfide bond with the fourth and last cysteine residues, and this particular structural feature may increase intermolecular aggregation of HBx protein and also result in diversity of HBx protein in solution after renaturation.

When the complete HBx protein or the truncated protein is expressed by using escherichia coli, the obtained target protein always exists in an inclusion body form, and the denaturation and renaturation are needed in the later purification, so that the natural form of the HBx protein is influenced to a certain extent. The X protein in trace soluble state can be obtained by utilizing a baculovirus insect expression system, most of the X protein is in an aggregation state, and the problems of denaturation and renaturation are also involved. At present, the bottleneck problem of X protein research is how to prepare high-purity X protein with correct conformation on a large scale, and the key to success is to select proper expression plasmids, expression hosts and add proper solubility-promoting labels. pET28a possesses T7 promoter, and this powerful and high specificity promoter is the first choice for prokaryotic expression through powerful design.

Disclosure of Invention

The invention aims to provide a method for preparing soluble hepatitis B virus X protein by a prokaryotic expression system, which avoids the influence of purified inclusion body protein, protease and inhibitor thereof on the unstable activity of the inclusion body protein and the protease, enhances the soluble expression of the HBx protein by fusing His and CL7 labels, and is convenient for the later purification and detection, thereby obtaining the high-purity HBx protein.

In order to achieve the above object, the present invention provides a method for purifying hepatitis B virus X protein, comprising the following steps:

s1, constructing a fusion expression vector of the hepatitis B virus X protein: respectively obtaining CL7, MBP and X gene sequences, inserting the fragments into a polyclonal enzyme cutting site of a pET28a plasmid by utilizing a T5 endonuclease-mediated recombination reaction to obtain expression vectors of pET28a-His-CL7-3C-HBx, pET28a-His-MBP-3C-HBx and pET28a-His-CL 7-MBP-3C-HBx;

s2, prokaryotic expression of recombinant expression protein: transforming the recombinant vector plasmid into BL21(DE3) competence, picking single colony from a flat plate after culture, carrying out small-amount induction expression, carrying out ultrasonic wave bacterial disruption, sampling, and analyzing the proportion of different fusion expression proteins in the supernatant of disrupted bacteria by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis);

s3, selecting induction conditions: optimizing expression conditions from induction temperature (18-37 ℃), inducer concentration (0.1-1 mmol/L) and induction time (12-20 h), ultrasonically crushing bacteria, sampling, and analyzing by SDS-PAGE to obtain optimal induction conditions;

s4, bulk induction: inoculating the cultured bacterial liquid into 1L LB liquid culture medium according to the proportion of 1:100, adding kanamycin to the final concentration of 100ug/ml, culturing at 18 ℃ and 220rpm, adding IPTG with the final concentration of 0.2mM when the OD600 value reaches about 0.6, and inducing for 16h at 18 ℃;

s5, protein supernatant acquisition: collecting the thallus cultured in step S4, re-suspending the thallus with lysis solution (lysis buffer: 20mM Tris-HCl, 500mM NaCl, pH 8.0), adding protease inhibitor (PMSF) to the final concentration of 0.1mM, ultrasonically breaking the thallus (400W,2/4S on/off,30min) in ice bath, high-speed centrifuging (10000rpm, 30min, 4 ℃) to collect supernatant, and filtering the supernatant with 0.02um filter membrane;

s6, Ni-NTA affinity chromatography: taking 8mL of NI-NTA, washing the equilibrium column by using lysine buffer with 3 times of the volume of the column bed, activating the NI-NTA resin by using buffer solution containing 20mM imidazole, and incubating the sample and the well-balanced column material at 4 ℃ overnight;

s7, gradient imidazole elution protein: washing the column with 3 column volumes of binding buffer, washing the column with gradient concentration imidazole (20mM, 50mM, 100mM, 200mM, 300mM), evaluating the optimal elution gradient, and collecting the eluate;

s8, ultrafiltration concentrated protein: analyzing the eluate collected in step S7 by SDS-PAGE, concentrating the high-purity eluate component with an ultrafiltration tube with a cut-off of 10KD, changing the eluate twice with a reaction buffer (50mM Tris-HCl, 150mM NaCl, pH 7.6) with a volume twice, and concentrating to 3 ml;

s9, HRV-3C protease enzyme digestion and secondary NI-NTA purification: performing enzyme digestion at 4 ℃ by using HRV 3C protease overnight, performing NI-NTA affinity chromatography on the enzyme-digested protein to remove a CL7 label and the HRV 3C protease, and analyzing the purity of the target protein by SDS-PAGE electrophoresis;

s10, protein verification: the immunogenicity was checked by Western Blot.

Preferably, the induction conditions in step S4 are IPTG concentration of 0.1-1mMol/L, induction temperature of 18-37 ℃, and induction expression time of 12-18 h.

Preferably, in step S6, the lysis solution includes 20mM Tris-HCl, 500mM NaCl, and pH 8.0.

Preferably, in step S7, the eluent used comprises a solution of 20-300 mM imidazole, 20mM Tris-HCl, 500mM NaCl, and has a pH of 8.0.

Preferably, in step S7, the column is washed 3 times by the 20 to 100mM imidazole solution and 2 times by the column volume by the 100 to 300mM imidazole solution.

Preferably, in step S8, the 3C enzyme digestion buffer comprises 50mM Tris-HCl, 150mM NaCl, pH 7.6.

An application of the hepatitis B virus X protein purification method is applied to the expression and purification of insoluble or insoluble protein products, including the structural research of HBx protein.

Therefore, the method for purifying the hepatitis B virus X protein comprises the steps of modifying the CE7 protein by a site-directed mutagenesis method, so that the CE7 protein loses nuclease activity and only retains the ability of combining with an inhibitory protein (IM7 protein) to obtain a mutant protein CL7, wherein the CL7 protein is a mutant of a nuclease structural domain which is derived from escherichia coli and has the ability of combining with DNA, and the CL7 protein has a certain dissolution promoting effect, can maintain the stability of the protein, improves the folding rate of the protein and improves the expression amount of a recombinant protein.

The invention constructs a pET28a-His-CL7-3C-HBx fusion expression system through molecular design, compares the dissolution promotion effect of a CL7 label and an MBP label on HBx in escherichia coli, and the result shows that the CL7-HBx fusion protein can be efficiently expressed in a soluble form in the escherichia coli, and finally obtains the hepatitis B virus X protein with high purity and high activity through the optimization of induction expression conditions, mass induction expression and two times of NI-NTA affinity chromatography.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is a flow chart of a method for purifying hepatitis B virus X protein according to the present invention;

FIG. 2 is a diagram showing the expression of different recombinant plasmids constructed according to the present invention in BL21(DE3) in small amounts; wherein, 1 is pET28a whole protein after no-load induced expression, 2 is pET28a-HBx induced expression supernatant protein, 3 is pET28a-HBx induced expression precipitation protein, 4 is pET28a-CL7-HBx induced expression supernatant protein, 5 is pET28a-CL7-HBx induced expression precipitation protein, 6 is pET28a-MBP-HBx induced expression supernatant protein, 7 is pET28a-MBP-HBx induced expression precipitation protein, 8 is pET28a-CL7-MBP-HBx induced expression supernatant protein, 9 is pET28a-CL7-MBP-HBx induced expression precipitation protein; lane M is protein Marker;

FIG. 3 is a graph showing the results of self-induced expression of 1mM IPTG fusion protein of the present invention under different temperature conditions; wherein lanes 1, 2 and 3 are total protein induced at 18 deg.C, supernatant protein after bacteria breaking, and precipitate protein after bacteria breaking; 4. lanes 5 and 6 are total protein, supernatant protein and precipitated protein after induction at 28 deg.C, respectively; 7. lanes 8 and 9 are total protein, supernatant protein and precipitated protein after induction at 37 deg.C, respectively; lane M is protein Marker;

FIG. 4 is a graph showing the results of quantitative induction expression of 1mM IPTG fusion protein of the present invention under different temperature conditions, in parallel with the data in FIG. 3;

FIG. 5 shows the results of 18 ℃ induced expression under different IPTG concentrations according to the present invention; wherein, lanes 1, 2 and 3 are respectively 0.1 mM/LIPTG-induced holoprotein, supernatant protein and precipitated protein, lanes 4, 5 and 6 are respectively 0.2 mM/LIPTG-induced holoprotein, supernatant protein and precipitated protein, lanes 7, 8 and 9 are respectively 0.4 mM/LIPTG-induced holoprotein, supernatant protein and precipitated protein, lanes 10, 11 and 12 are respectively 0.8 mM/LIPTG-induced holoprotein, supernatant protein and precipitated protein, lanes 13, 14 and 15 are respectively 1.0 mM/LIPTG-induced holoprotein, supernatant protein and precipitated protein, and lane M is protein Marker;

FIG. 6 shows the results of 18 ℃ induced expression quantification under IPTG conditions of different concentrations according to the present invention; wherein the data is the same as in FIG. 5 in parallel;

FIG. 7 shows the results of protein expression at different induction times according to the present invention; wherein, lanes 1, 2 and 3 are respectively whole protein, supernatant protein and precipitated protein induced for 12 h; 4. lanes 5 and 6 are total protein, supernatant protein and precipitated protein induced for 16h, respectively; 7. lanes 8 and 9 are total protein, supernatant protein and precipitated protein induced for 18h, respectively; lane M is protein Marker;

FIG. 8 shows the results of protein expression quantification at different induction times according to the present invention; the data are the same as in FIG. 6;

FIG. 9 is a purified view of a recombinant protein of the present invention (SDS-PAGE);

wherein, Lane 1 is the supernatant of the broken bacteria; lane 2 is flow through; lane 4 is 20mM imidazole wash; lane 5 is 50mM imidazole wash; lane 6 is 100mM imidazole wash; lane 7 is 200mM imidazole wash; lane 8 is 300mM imidazole wash; lane 9 is 300mM imidazole eluted twice wash; lane M is protein Marker;

FIG. 10 shows SDS-PAGE of the cleavage of HRV-3C protease of the present invention; wherein, lane 1 is the concentrated fusion protein; lane 2 is protein fluid after enzyme digestion with HRV 3C; lane 3 is the secondary Ni-NTA binding flow through; lane M is protein Marker;

FIG. 11 shows the result of Western Blot detection of the present invention, wherein the samples correspond to those in FIG. 9 one by one.

Detailed Description

The technical solution of the present invention is further illustrated by the accompanying drawings and examples.

Example one

Construction of recombinant X protein prokaryotic expression vector

(1) According to the HBx Gene sequence (Gene Bank access EU570072.1) published by Gene Bank, carrying out codon optimization design to synthesize the HBx Gene;

(2) carrying out reverse PCR amplification on a pET28a vector preserved in a laboratory to obtain a linear vector fragment;

(3) obtaining His-CL7, MBP and 3C-HBx linear fragments through PCR amplification;

(4) digesting the obtained three linear fragments by using T5 exonuclease, carrying out conventional transformation on products to escherichia coli DH5a competent cells, and selecting a single colony for culture to carry out bacteria liquid PCR and plasmid identification;

(5) after the recombinant plasmid is transformed into escherichia coli Bl21(DE3), the target recombinant engineering bacterium is obtained.

Example two

Induced expression and expression condition optimization of recombinant engineering bacteria

(1) Picking single colonies into 6ml of LB culture medium containing kanamycin respectively, culturing on a shaking table at 37 ℃ and 220rpm until OD600 is approximately equal to 0.6, adding 0.5mM of IPTG (isopropyl thiogalactoside) for induced expression for 4h, centrifuging and collecting 1ml of bacterial liquid after induction to prepare a protein sample, detecting the soluble expression of each label through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), wherein the result is shown in figure 2, the N-end fused CL7 label obviously promotes the solubility of HBx, the fused protein found in the precipitate is less than 30%, the N-end fused CL7-MBP label shows moderately enhanced solubility, the fused protein part is expressed in a soluble new form, about 50% is expressed in a new form of inclusion body, the fused MBP label does not improve the solubility of HBx, and almost all the fused proteins exist in a new form of inclusion body;

(2) inoculating the recombinant engineering bacteria transformed with pET28a-CL7-HBx into 6ml of liquid LB culture medium containing kanamycin, culturing on a shaker at 37 ℃ and 220rpm until the OD600 value is about 0.6, adding an inducer IPTG, continuously carrying out induction culture for 12 hours on the shaker at 18 ℃ and 28 ℃ and at 37 ℃ respectively at 220rpm, taking 2ml of bacterial liquid, carrying out ultrasonic bacteria breaking on the 2ml of bacterial liquid, respectively preparing samples of the whole bacterial liquid, supernatant after the bacteria breaking and precipitates after the bacteria breaking, and detecting the solubility of the recombinant protein expressed at different temperatures by SDS-PAGE electrophoresis, wherein the result is shown in a figure 3-4, an obvious band appears at about 35kDa of the induced whole protein, the size is consistent with the expected size, and the protein solubility is optimal under the induction condition of 18 ℃;

(3) the recombinant engineering bacteria transformed with pET28a-CL7-HBx are inoculated in 6ml of liquid LB culture medium containing kanamycin, when the liquid LB culture medium is cultured at 37 ℃ until the OD600 value is 0.6, final concentrations of 0.1mM, 0.2mM, 0.4mM, 0.8mM and 1mM IPTG are respectively added, after the liquid LB is induced for 12 hours by a shaking table at 18 ℃, the bacteria are collected, the bacteria are broken by ultrasonic waves, the whole bacteria liquid, the supernatant after the bacteria are broken and the sediment after the bacteria are respectively prepared into protein samples, and the expression of the recombinant protein is detected by SDS-PAGE electrophoresis, and the result is shown in figures 5-6, which shows that the induction condition of 0.2mM IPTG is optimal.

(4) Inoculating the recombinant engineering bacteria transformed with pET28a-CL7-HBx into 6ml of liquid LB culture medium containing kanamycin, culturing at 37 ℃ until the OD600 value is about 0.6, adding an inducer IPTG, respectively inducing at the shaking table of 220rpm of 18 ℃ for 12h, 16h and 20h, collecting thalli, ultrasonically breaking the thalli, respectively preparing protein samples from the whole bacterial liquid, supernatant after breaking the thalli and precipitates after breaking the thalli, detecting the expression of the recombinant protein through SDS-PAGE electrophoresis, wherein the result is shown in figures 7-8, and the result shows that the expression level of the soluble protein reaches the peak value after inducing for 16h, and the further extension of the induction time can not increase the dissolving capacity of the soluble protein;

(5) the optimal expression conditions are as follows: the temperature is 18 ℃, the induction time is 12h, and the concentration is 0.2mM IPTG;

EXAMPLE III

Two-step affinity purification of recombinant HBx protein

(1) Respectively inoculating recombinant engineering bacteria into 1L of liquid LB culture medium containing kanamycin, culturing at 37 ℃ until the OD600 value is about 0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.2mM, collecting the bacteria after 12h of shaking table induction at 18 ℃, re-suspending the bacteria by 100ml of lysate, adding protease inhibitor (PMSF) to the final concentration of 0.1mM, carrying out ultrasonic bacteria breaking in ice bath (400W, 30min), centrifugally collecting supernatant, and filtering by a 0.22um filter membrane;

(2) filling 8ml of nickel bead filler into a column by a wet method, and washing the column by pure water with the volume 10 times that of a column bed at the flow rate of 1.5 ml/min;

(3) the column was equilibrated with 5 bed volumes of lysate, 1.5 ml/min;

(4) combining the treated supernatant and the filtered liquid with nickel beads at 4 ℃ overnight, and passing through a column;

(5) washing with 3 times of column bed volume lysis solution Buffer for 3 times, and collecting eluate;

(6) washing for 3 times by using 3 times of column bed volume and 10-100 mM imidazole lysis solution, and collecting eluent;

(7) washing for 2 times by using a lysis solution containing 100-200 mM imidazole in 2 times of the volume of the column bed, and collecting an eluent;

(8) washing with 1 time of column bed volume 300mM imidazole lysate for 6 times, and collecting eluent;

(9) the purification results are shown in FIG. 9;

(10) collecting high-purity eluate, ultrafiltering, concentrating to obtain 2ml of target protein, and placing in 2 EP tubes;

(11) adding appropriate amount of HRV 3C protease, performing enzyme digestion at 4 deg.C overnight, adding the protein solution after enzyme digestion into 50ul nickel beads treated with 20mM imidazole in advance, combining at 4 deg.C for 1h, centrifuging at low speed (5000rpm,5min), carefully sucking the supernatant, quantitatively packaging and storing;

(12) the protease cleavage result is shown in FIG. 10;

(13) Western-Blot (HBx-specific antibody) analysis of fusion proteins

(14) The Western-Blot results are shown in FIG. 11.

Therefore, the purification method of the hepatitis B virus X protein avoids the influence of the renaturation of the purified inclusion body protein and the unstable activity of the protease and the inhibitor thereof, enhances the solubility of the HBx protein by fusing His and CL7 labels, and is convenient for affinity purification and detection in the later period, thereby obtaining the high-purity HBx protein.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.

Claims (7)

1. A method for purifying hepatitis B virus X protein is characterized by comprising the following steps:

s1, constructing a fusion expression vector of the hepatitis B virus X protein: respectively obtaining CL7, MBP and X gene sequences, inserting the fragments into a polyclonal enzyme cutting site of a pET28a plasmid by utilizing a T5 endonuclease-mediated recombination reaction to obtain expression vectors of pET28a-His-CL7-3C-HBx, pET28a-His-MBP-3C-HBx and pET28a-His-CL 7-MBP-3C-HBx;

s2, prokaryotic expression of recombinant protein: transforming the recombinant vector plasmid into BL21(DE3) competence, and performing induced expression after culture to obtain a target protein;

s3, optimizing recombinant protein expression conditions: optimizing expression conditions in 3 aspects of induction temperature (18-37 ℃), inducer concentration (0.1-1 mmol/L) and induction time (12-20 h);

s4, bulk induction: carrying out induced expression on the target protein after the cultured bacterial liquid is subjected to amplification culture;

s5, protein supernatant acquisition: collecting the thallus cultured in the step S4, re-suspending the thallus and a protease inhibitor (PMSF) by using a lysis buffer (lysis buffer), ultrasonically crushing the thallus in ice bath, collecting protein supernatant through high-speed centrifugation, and filtering and sterilizing the protein supernatant;

s6, His tag affinity chromatography: taking supernatant, adding NI-NTA agarose beads for combination, washing a balance column by using lysine buffer, activating the NI-NTA agarose beads by using 20-200mM imidazole, and incubating the sample and the balanced column material at 4 ℃ overnight;

s7, gradient imidazole elution protein: loading a sample on a column, collecting flow-through liquid, washing the column by combining buffer solution, washing column materials by gradient concentration imidazole, evaluating the optimal elution gradient, and collecting eluent;

s8, ultrafiltration concentrated protein: carrying out ultrafiltration concentration on the eluent collected in the step S7, then carrying out enzyme digestion on the buffer by using 3C enzyme, changing the solution, and concentrating;

s9, 3C enzyme digestion and secondary suspension of NI-NTA agarose beads: adding HRV 3C protease, performing enzyme digestion at 4 ℃ overnight, performing NI-NTA agarose affinity chromatography on the enzyme-digested protein, performing low-speed centrifugation, and carefully absorbing supernatant to obtain the required target protein;

s10, protein verification: the immunogenicity was checked by Western Blot.

2. The method of claim 1, wherein the purification step comprises: the induction condition in the step S4 is IPTG concentration of 0.1-1mMol/L, induction temperature of 18-37 ℃ and induction expression time of 12-18 h.

3. The method of claim 1, wherein the purification step comprises: in step S6, the lysate includes 20mM Tris-HCl, 500mM NaCl, and pH 8.0.

4. The method of claim 1, wherein the purification step comprises: in step S7, the eluent includes a solution of 20-300 mM imidazole, 20mM Tris-HCl, 500mM NaCl, and has a pH of 8.0.

5. The method of claim 1, wherein the purification step comprises: in step S8, the 3C enzyme digestion buffer includes 50mM Tris-HCl, 150mM NaCl, pH 7.6.

6. The method of claim 1, wherein the purification step comprises: in step S7, the column material is washed 3 times the column volume with 20-100 mM imidazole solution, and the column material is washed 3 times 2 times the column volume with 100-300 mM imidazole solution.

7. Use of a method for purifying hepatitis B virus X protein according to any of claims 1 to 6, characterized in that: the purification method is applied to expression and purification of insoluble or insoluble protein products, including structural research of HBx protein.

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