CN106754945B - Production method of toxin Tx4(6-1) tag-free recombinant protein - Google Patents

Abstract

The invention relates to a method for producing toxin Tx4(6-1) non-label recombinant protein, the gene provided by the invention is a DNA molecule shown by a sequence 1 in a sequence table; (1) by utilizing the gene sequence in the sequence table 1, the soluble high-level fusion expression of the toxin gene in escherichia coli can be realized; (2) cutting and removing the non-target protein from the fusion expressed soluble protein to obtain a recombinant protein which is completely identical to the natural mature Tx4(6-1) protein in amino acid sequence; (3) the obtained recombinant protein which is completely identical to the natural mature Tx4(6-1) protein in amino acid sequence has strong biological activity. The invention optimizes the DNA sequence of Tx4(6-1), screens expression strains and expression vectors, provides a method for efficiently producing active Tx4(6-1) recombinant protein, is beneficial to carrying out detailed research on the insect-resistant property and biological safety of the toxin protein, and has important significance for screening corresponding insect-resistant plants and high-toxicity transgenic biological pesticides.

Description

Production method of toxin Tx4(6-1) tag-free recombinant protein

Technical Field

The invention relates to the field of biotechnology utilization, in particular to production of an optimized coding gene and a recombinant protein of spider neurotoxin Tx4 (6-1).

Background

Neurotoxicity of insectsThe toxin is a kind of polypeptide neurotoxin which only acts on insect nervous system and has toxic and killing effects, mainly comes from scorpion, and secondly comes from spider. The action sites of the toxins with specific insect poisoning effect are various ion channels, the neurotoxins with high efficiency and insect specificity are mainly long-chain neurotoxins acting on sodium ion channels, the long-chain neurotoxins generally consist of 60-70 amino acids and have 2-4 pairs of intra-chain disulfide bonds. The neurotoxin from different species has no obvious conservative property in the amino acid composition, while the neurotoxin from the same species has high conservative property in the primary structure. The spider neurotoxin gene TX4(6-1) is derived from the brazil wandering spider: (Phoneutria nigriventer)The insect-specific neurotoxin has extremely good poisoning activity on agricultural pests. Insect neurotoxins have important potential value for controlling agricultural pests, but natural insect neurotoxins are low in toxic glands and difficult to extract, and often only a very small amount of pure product is available, which brings great difficulty for further study of the properties of these toxins. Therefore, it is an important way to research these toxins by separating a small amount of insect toxins, determining their amino acid composition, obtaining their full length mature genes, and expressing them in large amounts in vitro.

In the prior art, insect neurotoxin is constructed into an expression vector and then is transformed intoE. coliBL21(DE3) was expressed, but all obtained were inactive inclusion bodies, and by solubilization, denaturation, renaturation and purification under suitable conditions in vitro, only a trace amount of soluble protein was obtained per liter of medium, and the production was low; there is also a method of expressing the constructed expression vector in yeast, but the expression level is low and isolation and purification are difficult. At present, no way for efficiently producing Tx4(6-1) recombinant protein by modifying DNA sequence of Tx4(6-1) and optimizing Tx4(6-1) expression and purification method exists, so that the analysis and research on the insect-resistant property and biological safety of Tx4(6-1) are greatly influenced, and particularly the application of the recombinant protein in insect resistance is influenced, and therefore, a method for reducing cost, realizing mass production and having biological activity for insect neurotoxin Tx4(6-1) needs to be developed.

Disclosure of Invention

In view of the defects in the prior art, the invention aims to provide a coding gene of spider neurotoxin Tx4(6-1) and a production method of a recombinant protein thereof.

The invention provides a gene, which is a gene for coding a spider neurotoxin Tx4(6-1) protein and is a DNA molecule shown as a sequence 1 in a sequence table, wherein the reading frame of the coding gene comprises 147 nucleotides, the first 144 nucleotides code corresponding amino acids, and the last 3 nucleotides are stop codons. The protein translated into the sequence 1 in the sequence table comprises 48 amino acid residues and is a protein molecule shown as a sequence 2 in the sequence table.

The recombinant vector is specifically a recombinant vector for expressing the protein obtained by inserting the gene into an expression vector. The recombinant vector is particularly preferably B obtained by inserting the gene into an expression vector pET32amHI and HindIII, obtaining a recombinant vector for expressing the protein among enzyme cutting sites.

Primer pairs for amplifying the full length of the gene or any fragment thereof also belong to the protection scope of the invention. The nucleotide sequence of one primer in the primer pair is a sequence 3 in a sequence table, the nucleotide sequence of the other primer in the primer pair is a sequence 4 in the sequence table, and related enzyme cutting and connecting operations can be carried out on a target gene amplified by the primers with the sequences 3 and 4.

It is a second object of the present invention to provide a method for preparing a protein, comprising the steps of: a recombinant expression vector containing the gene of claim 1 is introduced into a host cell, preferably Escherichia coli, and the original vector for constructing the recombinant expression vector is preferably pET32 vector.

Further comprising the step of purifying the protein: cutting the expressed fusion protein by enterokinase, dialyzing in 5-10 mM Tris-HCl dialysis buffer solution with pH value of 6.5-6.6 by using a dialysis bag with molecular weight cutoff of 10kDa, adsorbing the dialyzed buffer solution by using a CM cation column, rinsing by using 10-30 mM NaCl solution, and eluting by using 100-150 mM NaCl to obtain the Tx4(6-1) recombinant protein with high purity.

The purified protein prepared by the method for preparing the protein also belongs to the protection scope of the invention.

The preferred method for preparing the protein specifically comprises:

s1: optimizing genes, constructing a prokaryotic expression vector and transforming: artificially synthesizing an optimized mature scorpion insect neurotoxin Tx4(6-1) gene, connecting the gene to an expression vector pET32 to obtain a recombinant expression vector pET32/Tx4(6-1), transforming the recombinant vector pET32/Tx4(6-1) into an escherichia coli TOP10 strain, and extracting a recombinant vector pET32/Tx4(6-1) from TOP 10; transferring the recombinant vector pET32/Tx4(6-1) into a host cell escherichia coli expression strain by a heat shock method, and screening an LB plate containing Amp resistance to obtain an escherichia coli expression strain transformant containing the recombinant vector pET32/Tx4 (6-1);

s2: expression and extraction of soluble Trx-Tx4(6-1) fusion protein: the E.coli expression strain transformant containing the recombinant vector pET32/Tx4(6-1) obtained in step S1 was cultured in LB liquid resistant medium at 37 ℃ to OD₆₀₀When the concentration is 0.5-0.8, adding IPTG (isopropyl thiogalactoside) with the concentration of 0.1-0.5 mM, inducing for 10-16 hours at the temperature of 18-25 ℃, then carrying out ultrasonic crushing, centrifuging and taking supernatant fluid to obtain a recombinant soluble fusion protein Trx-Tx4 (6-1);

s3: purification of Trx-Tx4(6-1) fusion protein: purifying the Trx-Tx4(6-1) fusion protein by using a nickel affinity chromatography column, rinsing the affinity chromatography column by using a pH 8.0 Tris-HCl solution containing 10mM imidazole, and eluting the fusion protein by using a pH 8.0 Tris-HCl solution containing 20-400 mM imidazole to obtain the Trx-Tx4(6-1) fusion protein with the purity of more than 90%;

s4: cleavage of Trx tag and purification of recombinant Tx4(6-1) protein: cutting the expressed fusion protein by enterokinase, dialyzing in 5-10 mM Tris-HCl dialysis buffer solution with pH value of 6.5-6.6 by using a dialysis bag with molecular weight cutoff of 10kDa, adsorbing the dialyzed buffer solution by using a CM cation column, rinsing by using 10-30 mM NaCl solution, and eluting by using 100-150 mM NaCl to obtain the Tx4(6-1) recombinant protein with high purity.

The technical scheme provided by the invention has the following advantages: firstly, the pET32 vector and an escherichia coli expression strain are utilized to realize the fusion of an expression product and a solubilizing factor Trx, and a large amount of soluble Trx-Tx4(6-1) fusion protein is obtained; secondly, in the expression system of the invention, the Trx-Tx4(6-1) fusion protein can be folded in a proper way and keeps a natural conformation; thirdly, a method for effectively purifying the active recombinant Trx-Tx4(6-1), quickly cutting the Trx and further purifying to obtain the Tx4(6-1) recombinant protein without any label is found out; fourthly, the Tx4(6-1) protein obtained by the expression of escherichia coli has high biological activity.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a schematic diagram of the construction of pET32/Tx4(6-1) vector in the examples of the present invention;

FIG. 2 is a SDS-PAGE result of soluble target proteins expressed by recombinant vectors pET16, pET20, pET28, pET36 and pET32 containing optimized Tx4(6-1) in the examples of the present invention;

FIG. 3 is a SDS-PAGE result of soluble Trx-Tx4(6-1) fusion protein expressed under optimized expression conditions in E.coli table of pET32/Tx4(6-1) vector containing optimized gene in the example of the present invention;

FIG. 4 is a graph showing SDS-PAGE detection results of a Trx-Tx4(6-1) fusion protein before purification and a Trx-Tx4(6-1) fusion protein purified using buffer C including imidazole at different concentrations according to an example of the present invention;

FIG. 5 is a SDS-PAGE detection result of the protein obtained by the enzyme digestion of the Trx-Tx4(6-1) fusion protein in the embodiment of the present invention;

FIG. 6 is a SDS-PAGE detection result of rTx4(6-1) recombinant protein in the example of the present invention;

FIG. 7 shows photographs taken of silkworms injected with rTx4(6-1) recombinant protein group at 1 hour after injection in the examples of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in examples are commercially available unless otherwise specified; in the examples,% is by mass unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.

The Escherichia coli expression strain, the vector amplification strain TOP10 and the expression vector pET32 are purchased from Invitrogen corporation of America. The specific sequences of the primers are shown as a sequence 3 and a sequence 4 in a sequence table, and the target genes amplified by the primers with the sequence 3 and the sequence 4 can also be subjected to related enzyme cutting and connection operations.

The formula of the culture medium is as follows:

1) LB liquid medium: 10 g of NaCl, 10 g of peptone, 5g of yeast extract and 1L of distilled water, and carrying out autoclaving and room-temperature storage;

2) LB/Amp plates: 10 g of NaCl, 10 g of peptone, 5g of yeast extract, 1L of distilled water and 15 g of agar powder, sterilizing under high pressure, cooling to below 70 ℃, adding 1mL of 100mg/mL Ampicillin (Ampicillin), mixing completely, pouring out, and storing at 4 ℃ in a dark place;

3) LB/Amp Medium: 10 g of NaCl, 10 g of peptone, 5g of yeast extract and 1L of distilled water, sterilizing at high pressure, cooling to below 70 ℃, adding 1mL of Ampicillin (100 mg/mL), mixing thoroughly, and storing at 4 ℃; LB liquid medium: 10 g of NaCl, 10 g of peptone, 5g of yeast extract and 1L of distilled water, and carrying out autoclaving and room-temperature storage;

4) 50 × TAE agarose gel electrophoresis buffer: 121 g of Tris alkali, 28.6 mL of glacial acetic acid, 50mL of 0.5 mol/L EDTA (pH 8.0), adding distilled water to a constant volume of 500 mL, and storing at room temperature;

5) 50 mg/mL ampicillin preservative solution: 0.5 g of ampicillin is dissolved by adding distilled water, the volume is determined to 10 mL, and the ampicillin is stored at minus 20 ℃ after split charging;

6) 5 XSDS-PAGE Loading buffer: 1.25 mL of 1M Tris-HCl (pH 6.8), 0.5 g of SDS, 25 mg of BPB and 2.5 mL of glycerol, adding deionized water for dissolving, then fixing the volume to 5 mL, subpackaging (about 500 mu L of each part), then storing at room temperature, adding 25 mu L of beta-mercaptoethanol into each part, and uniformly mixing;

7) 5 XSDS-PAGE running buffer: 15.1 g of Tris, 94 g of glycine and 5.0 g of SDS, adding about 800 mL of deionized water, fully stirring and dissolving, then fixing the volume to 1L, and storing at room temperature;

8) coomassie brilliant blue R-250 staining solution: adding 225 mL of methanol, 46 mL of glacial acetic acid and 225 mL of deionized water into the Coomassie brilliant blue R-2500.25 g, uniformly stirring, removing particulate matters by using filter paper, and storing at room temperature;

9) coomassie brilliant blue destaining solution: 50mL of glacial acetic acid, 150mL of methanol and 300 mL of deionized water are fully mixed and stored at room temperature.

Example one

The embodiment provides an optimized artificially synthesized Tx4(6-1) gene, the specific sequence of which is shown as sequence 1 in a sequence table, and the protein sequence corresponding to the gene is shown as sequence 2 in the sequence table. The sequence before optimization in this example was a DNA sequence provided in the NCBI database, which was a natural DNA of synthetic Tx4(6-1) neurotoxin, and the optimized DNA was optimized and synthesized according to the characteristics of E.coli expression. The NCBI database can only find the native protein sequence (GenBank accession No. 2108421A) of Tx4(6-1) without its corresponding nucleic acid sequence, thus optimizing the synthesisTx4(6-1)Genes do not have homologous DNA sequences.

The genes before and after optimization are connected to an escherichia coli expression vector pET series to obtain a recombinant vector, the recombinant vector verified by sequencing is respectively transformed into competent cells of an escherichia coli expression strain through heat shock, a corresponding resistant LB plate is coated, the culture is carried out in a constant temperature incubator at 37 ℃ for 12 hours, and transformants are screened, wherein the pET32/Tx4(6-1) vector construction is shown in figure 1, and figure 1 is a schematic diagram of pET32/Tx4(6-1) vector construction in the embodiment of the invention.

The expression of the target protein is not detected in the corresponding transformant of the expression strain by 0.5 mM IPTG induction at 37 ℃ by using the pET series recombinant vector containing the unoptimized natural Tx4(6-1) gene sequence as an expression vector, the SDS-PAGE result of the total bacterial protein is shown in figure 2, and the SDS-PAGE result of the target protein expressed by the pET series recombinant vector containing optimized Tx4(6-1) in the embodiment of the invention is shown in figure 2. pET series recombinant vectors containing optimized Tx4(6-1) gene sequences are used as expression vectors, corresponding expression bacteria transformants are induced at 37 ℃ by 0.5 mM IPTG, wherein the transformants taking pET18, pET20, pET28 and pET36 as the recombinant expression vectors do not detect the expression of target proteins, the transformants taking pET32 as the expression vectors detect the expression of the target proteins, the molecular weight of Tx4(6-1) protein is about 7 kDa, the pET32 vector fuses a Trx fragment with the size of about 18 kDa at the N-end of the target proteins, the size of Trx-Tx4(6-1) is about 25 kDa, and the expressed target proteins are shown by arrows.

Example two

This example provides a method for preparing a protein, comprising the steps of:

s1: optimizing genes, constructing a prokaryotic expression vector and transforming: an optimized mature scorpion insect neurotoxin Tx4(6-1) gene is artificially synthesized and is connected to an expression vector pET32 to obtain a recombinant expression vector pET32/Tx4(6-1), and the vector construction is shown in figure 1. The main vector construction steps are as follows:

(1) with BamH I and Hind III double restriction enzyme recombination vectorTx4(6-1)pUC, the target fragment PCP was obtained in the following reaction scheme (both the endonuclease and the buffer were purchased from Takara, Dalian company):

carrierTx4(6-1)/pUC 15 μL

10 XH buffer 5. mu.L

BamH Ⅰ 5U

HindⅢ 5U

Sterile water to 50 μ L

(2) With BamHI and HindIII double digestion of pET32 to obtain vector fragment, the reaction system is as follows (both the endonuclease and the buffer are purchased from the company of Dalian TAKARA):

vector pET 3215. mu.L

10 XH buffer 5. mu.L

BamHⅠ 5U

HindⅢ 5U

Sterile water to 50 μ L

(3) The target fragment and the vector fragment obtained in steps (1) and (2) were recovered by using a DNA gel retrieval kit purchased from Dalian TAKARA, and the detailed procedures were carried out according to the kit instructions.

(4) The target fragment and the vector recovered in the step (3) are connected by T4DNA ligase (purchased from TaKARA company of Dalian province), the target gene is accurately inserted into the reading frame of the vector, and the reaction system is as follows:

vector pET32 fragment 1. mu.L

Purpose fragment 3 uL

10 Xbuffer 1. mu.L

T4 ligase 0.5. mu.L

Sterile water to 10 μ L

The recombinant vector pET32 is combinedTx4(6-1)Transformed into the TOP10 strain of Escherichia coli, and the recombinant vector pET 32-Tx4(6-1)(ii) a Heat shock method was used to determine the location of the recombinant vector pET 32-Tx4(6-1)Transferred into a host cell escherichia coli expression strain, and screened by an Amp resistant LB plate to obtain a recombinant vector pET 32-Tx4(6-1)The E.coli expression strain transformant of (1).

S2: expression and extraction of soluble Trx-Tx4(6-1) fusion protein: the E.coli recombinant transformant containing the optimized gene pET32/Tx4(6-1) vector was cultured to OD in liquid LB medium at 37 ℃₆₀₀Is 0.6, howeverThen adding IPTG with the concentration of 0, 0.1, 0.5 and 1.0 mM respectively, inducing for 12 hours at the temperature of 18 ℃, 25 ℃ and 37 ℃, carrying out ultrasonic crushing on the collected thalli after induction, crushing power of 300W, crushing for 5 s, and spacing for 9 s, circulating for 90 times, and centrifuging to take supernatant fluid to obtain the recombinant soluble fusion protein Trx-Tx4 (6-1).

S3: purification of Trx-Tx4(6-1) fusion protein: after amplification culture and induction with 0.1 mM IPTG at 18 ℃ for 12 hours, collecting thalli of expression bacteria after IPTG induction expression, suspending the thalli in 50ml of buffer solution A (containing 20 mM Tris-HCl, 100 mM NaCl, 10mM imidazole and 1 mM protease inhibitor PMSF, pH 8.0), then crushing with an ultrasonic crusher with the crushing power of 300W for 5 s and the gap of 9 s, and circulating for 90 times; centrifuging the crushed bacterial liquid at the temperature of 4 ℃ at 30000 g for 15 min; adding the supernatant obtained by centrifugation into a nickel affinity chromatography column pre-balanced by a buffer solution A; rinsing the protein purification column with 100 ml of buffer solution B (containing 20 mM Tris-HCl, 100 mM NaCl, 10mM imidazole and 0.5-1.0% Triton X-114, pH 8.0, pre-cooling to 0-4 ℃ in an ice bath, respectively adding buffer solution C (containing 20 mM Tris-HCl, 100 mM NaCl, pH 8.0) containing 20-400 mM imidazole, and eluting the protein to obtain Trx-Tx4(6-1) fusion protein with the purity of more than 90%.

S4: cleavage of Trx tag and purification of recombinant Tx4(6-1) protein: taking 1 mg of Trx-Tx4(6-1) fusion protein, adding 2-3U of recombinant calf enterokinase, and carrying out enzyme digestion at 25 ℃ for 6 hours for sampling. And (3) after enzyme digestion, dialyzing the dialyzed buffer solution in 5-10 mM Tris-HCl dialysis buffer solution with the pH value of 6.5-6.6 by using a dialysis bag with the molecular weight cutoff of 10kDa, adsorbing the dialyzed buffer solution by using a CM cation column, rinsing the dialyzed buffer solution by using 10-30 mM NaCl solution, and eluting the dialyzed buffer solution by using 100-150 mM NaCl to obtain Tx4(6-1) recombinant protein with the purity of more than 95%, wherein 10 mg of high-purity Tx4(6-1) recombinant protein can be finally obtained from 1L of escherichia coli.

In addition, SDS-PAGE sample buffer was added to the supernatant obtained in step S2, and the soluble protein was analyzed. Soluble Trx-Tx4(6-1) fusion proteins were obtained at IPTG concentrations of 0.1 mM, 0.5 mM and 1.0 mM at 18 deg.C, 25 deg.C and 37 deg.C, as shown in FIG. 3, and FIG. 3 is a graph of SDS-PAGE detection of soluble Trx-Tx4(6-1) fusion proteins expressed from E.coli vectors containing optimized genes pET32/Tx4(6-1) in the examples of the present invention. Wherein, at the temperature of 18 ℃, the soluble Trx-Tx4(6-1) can be expressed at high level in the test concentration range; higher levels of soluble Trx-Tx4(6-1) expression at IPTG concentrations of 0.1 mM to 0.5 mM at 25 ℃. In order to save cost and shorten production period, the IPTG with the induction temperature of 18-25 ℃ and the induction temperature of 0.1-0.5 mM is preferably adopted for induction expression.

For step S3, the results show that the recombinant protein with purity of 90% can be obtained by elution with imidazole buffer C containing 100-400 mM, the molecular weight of the fusion protein is about 25 kDa, the optimization of the purification process of the expression product soluble Trx-Tx4(6-1) is shown in FIG. 4, and FIG. 4 is a graph of the SDS-PAGE detection results of the Trx-Tx4(6-1) fusion protein before purification and the Trx-Tx4(6-1) fusion protein obtained by purification with buffer C containing imidazole with different concentrations in the examples of the present invention.

The cleavage effect was analyzed by SDS-PAGE in step S4, and the results are shown in FIG. 5. Performing enzyme digestion for 4 hours to obtain a cut rTx4(6-1) protein (target protein is shown by an arrow), dialyzing the dialyzed bag with the molecular weight cutoff of 10kDa in a 5-10 mM Tris-HCl dialyzing buffer solution with the pH value of 6.5-6.6, adsorbing the buffer solution on the outer side of the dialyzed bag by using a CM cation column, rinsing the buffer solution by using a 10-30 mM NaCl solution, and eluting the buffer solution by using 100-150 mM NaCl to obtain the high-purity rTx4(6-1) recombinant protein without a label. rTx4(6-1), the results of SDS-PAGE detection of the recombinant protein are shown in FIG. 6.

The purified recombinant protein rTx4(6-1) was analyzed for biological activity, and the specific analysis method and results are as follows.

The experimental method comprises the following steps: the purified Trx-Tx4(6-1) fusion protein (group A) and the purified rTx4(6-1) protein (group B) were diluted to 2 mg/ml, 30 silkworms of 4 years old were injected at 10. mu.g/g body weight, and the poisoning symptoms of silkworms of 4 years old were observed using silkworms of 4 years old injected with PBS of the same volume (group C) as controls.

The experimental results are as follows: as a result, it was found that silkworms injected with the purified rTx4(6-1) protein immediately exhibited strong neurotoxin intoxication symptoms and all silkworms died within 24 hours, silkworms injected with the purified Trx-Tx4(6-1) fusion protein exhibited no significant nervous system intoxication symptoms, and the mortality rate was 10% within 24 hours, whereas none of the silkworms of the control group injected with the same volume of PBS exhibited intoxication symptoms. The results observed after 1 injection hour in the control group in which purified rTx4(6-1) silkworms and PBS were injected are shown in fig. 7, in which the injection group showed significant contraction symptoms. The biological activity detection result shows that the rTx4(6-1) protein prepared by the technical scheme of the invention has strong biological activity. The specific results are shown in table 1 below:

table 1A, B, C and group D biological activity assay results (n = 3).

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments within the scope of the present invention without departing the essence of the corresponding embodiments from the scope of the embodiments of the present invention, which should be covered in the claims and the specification of the present invention.

SEQUENCE LISTING

<110> college of bosom

<120> method for producing toxin Tx4(6-1) non-tag recombinant protein

<130> 2016-12-15-

<160> 4

<170> PatentIn version 3.3

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Claims (4)

1. The recombinant vector is characterized in that a gene with a nucleotide shown as a sequence 1 is inserted into an expression vector to obtain the recombinant vector for expressing a protein shown as a sequence 2, and the expression vector is a pET32 vector.

2. A recombinant bacterium constructed from the recombinant vector according to claim 1, wherein the recombinant bacterium is obtained by introducing the recombinant vector according to claim 1 into Escherichia coli.

3. A method for producing a protein, characterized in that the recombinant bacterium of claim 2 is cultured and expressed in LB medium.

4. The method for producing a protein according to claim 3, wherein: the method further comprises the step of purifying the protein: cutting the expressed fusion protein by enterokinase, dialyzing in 5-10 mM Tris-HCl dialysis buffer solution with pH value of 6.5-6.6 by using a dialysis bag with molecular weight cutoff of 10kDa, rinsing the dialyzed buffer solution with 10-30 mM NaCl solution after adsorbing by using a cation column, and eluting by using 100-150 mM NaCl to obtain the Tx4(6-1) recombinant protein with high purity.

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