CN104152426A - Preparation method of T5 exonuclease in escherichia coli - Google Patents

Abstract

The invention provides a preparation method of T5 exonuclease in escherichia coli, and belongs to the field of biochemistry. According to the preparation method provided by the invention, T5 exonuclease gene and an enterokinase restriction site are cloned onto a pGEX vector containing a GST tag through vector construction, under a low temperature and low concentration IPTG condition, the T5 exonuclease fusion protein expression is induced, and the protein expression index of protein reaches 15 percent. Through GST affinity chromatography, EK digestion and ion exchange chromatographic purification, T5 exonuclease without any tags is obtained, and the specific activity is about 8000 units/mg.

Description

The preparation method of a kind of T5 exonuclease in intestinal bacteria

Technical field

The present invention relates to expression and the purification process of a kind of T5 exonuclease (T5 Exonuclease) in intestinal bacteria, belong to biochemical field.

Background technology

Exonuclease (exonucleautomotive service engineers) is in hydrolase nucleic acid, to have from the end of molecular chain be hydrolyzed phosphodiester bond in turn and generate the enzyme of mononucleotide effect, always to single nucleotide chain effect, in shearing modification reaction, work, corresponding with endonuclease.3 ' the end that can roughly be divided into hydrolysis phosphodiester bond generates the enzyme of 5 '-mononucleotide, and hydrolysis 5 ' end generates the enzyme of 3 '-mononucleotide.The former has snake venom phosphodiesterase and Escherichia coli nucleic acid excision enzyme I, II and III etc.; The latter has spleen phosphodiesterase, Lactobacterium acidophilum (Lac-tobacillus acidophilus) nuclease.In these enzymes, can also distinguish the enzyme that starts to cut off and single stranded DNA or double-stranded DNA are had specific action from 3 ' end of molecular chain or 5 ' end.Can utilize the difference of these enzymic hydrolysis modes to carry out analysis of nucleic acids structure.The E.coli bacterial strain of the self-contained T5 phage of T5 exonuclease purifying D15 gene plasmid, is the one of exonuclease, and this enzyme can be along 5 ' → 3 ' direction degradation of dna.It can be from the initial digestion of 5 ' end, also can be from the nicking of linearity or circular double stranded DNA or the initial digestion of indentation, there, but the superhelix double-stranded DNA of can not degrading.T5 exonuclease also has single stranded DNA endonuclease activity.These characteristics are often used in DNA digestion and gene clone T5 exonuclease.Along with popularizing of gene clone technology, T5 exonuclease market demand is increasing, and extractive technique complexity, the cost of natural T5 exonuclease are high, productive rate is low.

Summary of the invention

The object of the invention is for solving above-mentioned difficulties, a kind of restructuring T5 exonuclease (T5 Exonuclease) solubility expression and purification process in intestinal bacteria are provided, the present invention has done codon optimized by T5 Exonuclease gene, by vector construction, T5 exonuclease gene and enteropeptidase restriction enzyme site are cloned in pGEX carrier, under the inductive condition of 16 DEG C, the expression amount of T5 exonuclease can reach 15%, and solubility reaches 95%.Finally, cut the not T5 exonuclease with any label of purifying by GST label purifying and enteropeptidase enzyme.

The present invention realizes with following technical scheme: a kind of restructuring T5 exonuclease, the nucleotide sequence of the T5 exonuclease it is characterized in that is as shown in sequence table SEQ ID NO:1.

Its preparation method comprise the steps: by vector construction by codon optimized T5 exonuclease gene clone in pGEX carrier, under cold condition by with GST amalgamation and expression, improve expressing quantity and solubility; T5 exonuclease gene order is as shown in sequence table SEQ ID NO:1; T5 exonuclease and GST amalgamation and expression in Host Strains, amalgamation and expression albumen is GST-T5 Exonuclease; Amalgamation and expression albumen contains GST label, with GST pillar purified fusion protein.

Further, vector construction step is specially following steps:

Design of primers is as follows:

Forward primer used is:

5’-CATG GGATCCGACGACGACGACAAGATGAGCAAATCGTGGGGGAAATTCAT-3’；

Reverse primer used is:

5’-CATG CTCGAGTCACTGTTCTGCAATTTCGA-3’；

In forward primer, there is the sequence of restriction enzyme site and the coding enteropeptidase recognition site DDDDK of a BamH I, in reverse primer, have the restriction enzyme site of an Xho I; To synthesize T5 exonuclease gene as template, with the method amplification T5 exonuclease encoding sequence of PCR, the condition of PCR reaction is: 94 DEG C of thermally denatures one minute, and 55 DEG C of annealing one minute, 72 DEG C are extended 90 seconds, carry out altogether 25 circulations; The PCR product of pGEX and purifying adopts BamH I and Xho I to carry out double digestion, then PCR endonuclease bamhi is reclaimed to the multiple clone site of inserting pGEX; PGEX is a prokaryotic expression carrier that merges GST-tag at N end; Recombinant expression vector pGEX-T5 exonuclease is carried out to DNA sequencing; Analyze the exactness of its reading frame and encoding sequence;

Further, amalgamation and expression step is specially following steps:

T5 exonuclease fusion protein expression vector pGEX-T5 exonuclease is proceeded to expressive host bacterium E.coli BL21 (DE3), and picking mono-clonal is seeded to the LB liquid nutrient medium of the fresh penbritin containing 50mg/l; Treat that bacterium grows to OD ₆₀₀be 0.6 left and right, adding final concentration is the expression of the IPTG induction T5 exonuclease of 0.4mM; 16 DEG C of inductions were collected thalline after 12 hours.

Further, purified fusion protein step is specially following steps:

Collect the cell of one liter of fermented liquid, and resuspended with the buffer A of 40ml ice bath, the formation of described buffer A is 20mM Tris-HCl, 50mM NaCl, 0.5% Triton-X100,10% glycerine, ultrasonic degradation cell, centrifuging and taking supernatant, supernatant is by the GST affinity column of buffer A pre-equilibration, and loading is complete fully washs to baseline with buffer A, then use buffer B wash-out target protein, the formation of described buffer B is 50mM Tris-HCl, 20mM Reduced Glutathione, 10% glycerine;

In connection with cutting with EK enzyme after the dialysis of GST-T5 exonuclease, according to EK (mg): the ratio of target protein (mg)=1: 10000 adds EK in fusion rotein solution and starts endonuclease reaction, enzyme tangent condition is 4 DEG C, 16 hours, enzyme is cut solution and is collected effluent liquid by GST affinity column, and effluent liquid is further through strong anion exchange absorbing chromatography Q-Sepharose purifying.

Advantage of the present invention is: by vector construction, T5 exonuclease gene and enteropeptidase restriction enzyme site are cloned in the pGEX carrier containing GST label, under low temperature, lower concentration IPTG condition, induce expressing fusion protein.The purifying that makes T5 exonuclease that adds of the GST label containing in pGEX carrier in addition, and enteropeptidase restriction enzyme site is simplified, efficiently more.

Brief description of the drawings

Below in conjunction with drawings and Examples, the invention will be further described:

Figure 1A is the plasmid map of recombinant expression vector pGEX-T5 exonuclease;

Figure 1B is that the SDS-PAGE of restructuring GST-T5 exonuclease protein expression analyzes;

Swimming lane 1: molecular weight of albumen standard (kD); Lanes2 and 3: be respectively the pGEX-T5 exonuclease that does not add IPTG induction and the full bacteria culture fluid of pGEX-T5 exonuclease that adds IPTG induction;

Fig. 2 A is GST column purification GST-T5 exonuclease fusion rotein;

Swimming lane 1: the ultrasonic supernatant of great expression; Swimming lane 2:GST purification media is passed; Swimming lane 3:buffer A washing; Swimming lane 4:Buffer B wash-out fusion rotein; Swimming lane 5: molecular weight of albumen standard (KD);

Fig. 2 B is that enteropeptidase enzyme is cut fusion rotein, and collects T5 exonuclease albumen;

Swimming lane 1: molecular weight of albumen standard (KD); Swimming lane 2: enzyme is cut GST-T5 exonuclease fusion rotein; Swimming lane 3:GST purification media is passed, without the T5 exonuclease of label;

Fig. 3 is T5 exonuclease vitality test;

Swimming lane 1: nucleic acid molecular weight standard (bp); Swimming lane 2: not enzyme-added; Swimming lane 3:0.2U T5 exonuclease (NEB company); Swimming lane 4:0.2U T5 exonuclease (Fig. 2 B purifying); Swimming lane 5:1U T5 exonuclease (NEB company); Swimming lane 6:1U T5 exonuclease (Fig. 2 B purifying)

Embodiment

The present invention is by the structure of restructuring T5 exonuclease prokaryotic expression carrier pGEX-T5 exonuclease, success by codon optimized T5 exonuclease gene clone to the downstream of GST encoding sequence and with it in same reading frame, between GST and T5 exonuclease, add the restriction enzyme site of enteropeptidase, built fusion rotein pGEX-T5 exonuclease prokaryotic expression carrier, sequencing result shows that reading frame and DNA sequence dna are all correct.By contrast experiment, find under low temperature, low IPTG inductive condition, the expression amount 15% of pGEX-T5 exonuclease albumen, and solubility reaches 95%.Cut and GST affinity chromatography is received through albumen by GST affinity chromatography, EK enzyme, obtained the not T5 exonuclease with any affinity tag.GST affinity chromatography has been removed most thalline foreign proteins, after cutting with enteropeptidase enzyme, will not be with the amino acid whose T5 exonuclease of any redundancy and label to separate, finally remove the DNA of EK and trace with ion-exchange, in one liter of fermented liquid, obtained the T5 exonuclease (purity approximately 98%) of about 8mg.12% SDS-PAGE analyzes and shows, the T5 exonuclease of purifying conforms to theoretical molecular 31.1kD.The T5 exonuclease of purifying shows and the similar activity of commercialization enzyme (NEB company), and specific activity is about 8000units/mg.

Below by preferred embodiment, the present invention is done to more detailed discussion.Experimental technique in following experiment, if without specified otherwise, all belongs to ordinary method.

1, the structure of restructuring T5 exonuclease prokaryotic expression carrier pGEX-T5 exonuclease:

To synthesize T5 exonuclease gene as template, with the method amplification T5 exonuclease encoding sequence of PCR,

Forward primer used is:

5’-CATG GGATCCGACGACGACGACAAGATGAGCAAATCGTGGGGGAAATTCAT-3’，

Reverse primer is: 5 '-CATG cTCGAGtCACTGTTCTGCAATTTCGA-3 ';

In forward primer, there is the sequence of restriction enzyme site and the coding enteropeptidase recognition site DDDDK of a BamH I, in reverse primer, have the restriction enzyme site of an Xho I.The condition of PCR reaction is: 94 DEG C of thermally denatures one minute, and 55 DEG C of annealing one minute, 72 DEG C are extended 90 seconds, carry out altogether 25 circulations.The PCR product of pGEX and purifying adopts BamH I and Xho I to carry out double digestion, then PCR endonuclease bamhi is reclaimed to the multiple clone site of inserting pGEX.PGEX is a prokaryotic expression carrier that merges GST-tag at N end.Recombinant expression vector pGEX-T5 exonuclease is carried out to DNA sequencing, analyze the exactness of its reading frame and encoding sequence.

According to the step described in method, by codon optimized T5 exonuclease gene clone to the downstream of GST encoding sequence and with it in same reading frame, for the ease of discharging the also T5 exonuclease of purifying wild-type, the present invention designs and adds the restriction enzyme site of enteropeptidase between GST-tag and T5 exonuclease gene, recombinant plasmid is through sequence verification, successfully build the prokaryotic expression carrier of fusion rotein pGEX-T5 exonuclease, reading frame and DNA sequence dna are all correct, and its sketch is referring to Figure 1A.

The expression of 2.pGEX-T5 exonuclease:

T5 exonuclease fusion protein expression vector pGEX-T5 exonuclease is proceeded to expressive host bacterium E.coli BL21 (DE3), and picking mono-clonal is seeded to fresh LB liquid nutrient medium (containing the penbritin of 50mg/l).Treat that it is 0.6 left and right that bacterium grows to OD600, adding final concentration is the expression of the IPTG induction T5 exonuclease of 0.4mM.16 DEG C of inductions were collected thalline after 12 hours, tropina sample after treatment runs 12%SDS-PAGE, use UVP white/ultraviolet transilluminator to carry out sweep record to the gel of Coomassie brilliant blue dyeing, adopt specialty analysis software Grab-it 2.5 and Gelwork analysis purposes albumen to account for the ratio of bacterial protein.Herein, solubility is defined as: target protein/general purpose albumen × 100% of solubility.After IPTG induction, occurred a protein band that is about 64kD, and theoretical molecular size is identical, through gel image scanning gray analysis, T5 exonuclease fusion rotein accounts for 15% left and right (Figure 1B) of bacterial protein,

3, the purifying of albumen

Collect the cell of one liter of fermented liquid, and with bufferA (20mM Tris-HCl, the 50mM NaCl of 40ml ice bath, 0.5% Triton-X100,10% glycerine, pH8.0 is resuspended, ultrasonic degradation cell, centrifuging and taking supernatant, supernatant is by the GST affinity column of buffer A pre-equilibration, and loading is complete fully washs to baseline with buffer A, then use buffer B (50mM Tris-HCl, 20mM Reduced Glutathione, 10% glycerine, pH8.0) wash-out target protein.

In connection with cutting with EK enzyme after the dialysis of GST-T5 exonuclease, according to EK (mg): the ratio of target protein (mg)=1: 10000 adds EK in fusion rotein solution and starts endonuclease reaction, enzyme tangent condition is 4 DEG C, 16 hours, enzyme is cut solution and is collected effluent liquid by GST affinity column, and effluent liquid is further through strong anion exchange absorbing chromatography Q-Sepharose purifying.Enzyme after purifying adopts Micro BCA test kit quantitatively and gel gray scale scanning is analyzed.

Cut and ion exchange chromatography purifying by GST affinity chromatography, EK enzyme, obtained the not T5 exonuclease with any affinity tag of wild-type.GST affinity chromatography has been removed most thalline foreign proteins, cut with enteropeptidase enzyme, to not be with the amino acid whose T5 exonuclease of any redundancy and GST label to separate, finally remove the DNA of EK and trace by ion exchange chromatography, finally from one liter of fermented liquid, obtained the wild-type T5 exonuclease (purity approximately 95%) of about 8mg.12%SDS-PAGE analyzes and shows, the T5 exonuclease of purifying conform to theoretical molecular 31.6kD (Fig. 2 B).

The activity test method of 4.T5 exonuclease

1 unit T5 exonuclease refers in 50 μ l reaction systems, 37 DEG C of reactions 30 minutes, can be from double-stranded DNA substrate catalysis produce the needed enzyme amount of acid-solubility deoxyribonucleotide of 1nmol.

As shown in Figure 3, the T5 exonuclease of purifying shows and the similar activity of commercialization enzyme (NEB company), and specific activity is about 8000units/mg.

Claims (5)

1. the preparation method of T5 exonuclease in intestinal bacteria, is characterized in that the nucleotide sequence of described T5 excision enzyme is as shown in sequence table SEQ ID NO:1.

2. the preparation method of a kind of T5 exonuclease as claimed in claim 1 in intestinal bacteria, it is characterized in that, comprise the steps: by vector construction by codon optimized T5 exonuclease gene clone in pGEX carrier, under cold condition by with GST amalgamation and expression, improve expressing quantity and solubility; Described T5 exonuclease gene order is as shown in sequence table SEQ ID NO:1; T5 exonuclease and GST amalgamation and expression in Host Strains, amalgamation and expression albumen is GST-T5 Exonuclease; Described amalgamation and expression albumen contains GST label, with GST pillar purified fusion protein.

3. the preparation method of a kind of T5 exonuclease according to claim 2 in intestinal bacteria, is characterized in that described vector construction step is specially following steps:

Design of primers is as follows:

Forward primer used is:

5 ' CATG gGATCCgACGACGACGACAAGATGAGCAAATCGTGGGGGAAATTCAT-3 '; Underscore part is the restriction enzyme site of BamH I;

Reverse primer used is:

5 '-CATG cTCGAGtCACTGTTCTGCAATTTCGA-3 '; Underscore part is the restriction enzyme site of Xho I;

In forward primer, there is the sequence of restriction enzyme site and the coding enteropeptidase recognition site DDDDK of a BamH I, in reverse primer, have the restriction enzyme site of an Xho I; To synthesize T5 exonuclease gene as template, with the method amplification T5 exonuclease encoding sequence of PCR, the condition of PCR reaction is: 94 DEG C of thermally denatures one minute, and 55 DEG C of annealing one minute, 72 DEG C are extended 90 seconds, carry out altogether 25 circulations; The PCR product of pGEX and purifying adopts BamH I and Xho I to carry out double digestion, then PCR endonuclease bamhi is reclaimed to the multiple clone site of inserting pGEX; PGEX is a prokaryotic expression carrier that merges GST-tag at N end; Recombinant expression vector pGEX-T5 exonuclease is carried out to DNA sequencing; Analyze the exactness of its reading frame and encoding sequence.

4. the preparation method of a kind of T5 exonuclease according to claim 2 in intestinal bacteria, is characterized in that described amalgamation and expression step is specially following steps:

T5 exonuclease fusion protein expression vector pGEX-T5 exonuclease is proceeded to expressive host bacterium E.coli BL21 (DE3), and picking mono-clonal is seeded to the LB liquid nutrient medium of the fresh penbritin containing 50mg/l; Treat that it is 0.6 left and right that bacterium grows to OD600, adding final concentration is the expression of the IPTG induction T5 exonuclease of 0.4mM; 16 DEG C of inductions were collected thalline after 12 hours.

5. the preparation method of a kind of T5 exonuclease according to claim 2 in intestinal bacteria, is characterized in that described purified fusion protein step is specially following steps:

Collect the cell of one liter of fermented liquid, and resuspended with the buffer A of 40ml ice bath, the formation of described buffer A is 20mMTris-HCl, 50mM NaCl, 0.5% Triton-X100,10% glycerine, ultrasonic degradation cell, centrifuging and taking supernatant, supernatant is by the GST affinity column of buffer A pre-equilibration, and loading is complete fully washs to baseline with buffer A, then use buffer B wash-out target protein, the formation of described buffer B is 50mM Tris-HCl, 20mM Reduced Glutathione, 10% glycerine;

In connection with cutting with EK enzyme after the dialysis of GST-T5 exonuclease, according to EK (mg): the ratio of target protein (mg)=1: 10000 adds EK in fusion rotein solution and starts endonuclease reaction, enzyme tangent condition is 4 DEG C, 16 hours, enzyme is cut solution and is collected effluent liquid by GST affinity column, and effluent liquid is further through strong anion exchange absorbing chromatography Q-Sepharose purifying.