CN104178467A - Recombinant T4 bacteriophage polynucleotide kinase (T4 PNK) and preparation method thereof - Google Patents

Recombinant T4 bacteriophage polynucleotide kinase (T4 PNK) and preparation method thereof Download PDF

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CN104178467A
CN104178467A CN201410361153.9A CN201410361153A CN104178467A CN 104178467 A CN104178467 A CN 104178467A CN 201410361153 A CN201410361153 A CN 201410361153A CN 104178467 A CN104178467 A CN 104178467A
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polynucleotide kinase
pnk
gst
expression
recombinant
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孙启明
张亚
徐卫国
陈远
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The invention relates to expression and purification of T4 bacteriophage polynucleotide kinase (T4 PNK) and particularly relates to soluble expression and a purification method of the T4 PNK, belonging to the field of biochemistry. According to the invention, a T4 PNK gene is cloned into a pGEX vector through vector construction, the T4 polynucleotide kinase and GST undergo fused expression under the condition of low temperature, then, the protein expression level reaches 15%, and the solubility reaches 95%.

Description

A kind of recombinant T 4 bacteriophage polynucleotide kinase and preparation method thereof
Technical field
The present invention relates to a kind of T4 phage polynucleotide kinase (T4 bacteriophage polynucleotide kinase, T4PNK) expression and purifying, be particularly related to solubility expression and the purification process of a kind of T4 phage polynucleotide kinase (T4 bacteriophage polynucleotide kinase, T4 PNK).Belong to biochemical field.
Background technology
T4 phage polynucleotide kinase (T4 bacteriophage polynucleotide kinase, T4 PNK), a kind of polynucleotide 5 ' hydroxyl kinases, can catalysis ATP? position phosphate group shifts to strand or double-stranded DNA, RNA, oligonucleotide or with 5 ' hydroxyl of the mononucleotide of 3 ' phosphate group.Can other NTP also produce identical reaction: 5 '-OH+NTP? 5 '-P+NDP.Does T4Polynucleotide Kinase have 3 ' phosphate esterase active simultaneously, the dephosphorylation of polynucleotide that can catalysis 3 ' phosphorylation: 3 '-P? 3 '-OH+Pi.T4 phage polynucleotide kinase is widely used, as 5 ' end mark of oligonucleotide, DNA or RNA, as the probe of Southern, Northern, EMSA etc., the marker of gel electrophoresis, DNA sequencing primer, PCR primer etc.; Make 5 of oligonucleotide, DNA or RNA ' end phosphorylation, guarantee that follow-up ligation carries out smoothly.As conventional molecular biology product, the T4 phage polynucleotide kinase huge market demand, recombinant T 4 bacteriophage polynucleotide kinase expression amount in pET system of genetic engineering technique clone is high at present, but easily forms inclusion body, after purifying, can only obtain a small amount of activated protein.
Summary of the invention
The object of the invention is the weak point for overcoming above-mentioned technology, a kind of recombinant T 4 bacteriophage polynucleotide kinase solubility expression and purification process in intestinal bacteria are provided.The present invention by vector construction by the gene clone of T4 phage polynucleotide kinase in pGEX carrier, T4 phage polynucleotide kinase and GST amalgamation and expression under cold condition, T4 phage polynucleotide kinase expressing quantity can reach 15%, and solubility reaches 95%.
The present invention realizes with following technical scheme: a kind of recombinant T 4 bacteriophage polynucleotide kinase, the nucleotide sequence of the T4 phage polynucleotide kinase it is characterized in that is as shown in sequence table SEQ IF NO:1.
Its preparation method comprise the steps: by vector construction by codon optimized T4 phage polynucleotide kinase gene clone in pGEX carrier, under cold condition by with GST amalgamation and expression, improve expressing quantity and solubility; T4 phage polynucleotide kinase gene order is as shown in sequence table SEQ ID NO:1; T4 phage polynucleotide kinase and GST amalgamation and expression in Host Strains, amalgamation and expression albumen is GST-T4 PNK; 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 gGATCCaTGAAGAAAATCATT-3 '; Underscore part is the restriction enzyme site of BamHI;
Reverse primer used is: 5 '-CATG cTCGAGtCAAAAGTCCCCACTAG-3 '; Underscore part is the restriction enzyme site of XhoI;
Taking the T4 PNK that synthesizes as template, with the method amplification T4 PNK encoding sequence of PCR, PCR reaction conditions is: 94 DEG C of thermally denatures 1 minute, and 55 DEG C of annealing 1min, 72 DEG C are extended 90 seconds, carry out altogether 25 circulations; The PCR product of purifying carries out double digestion with BamHI and XhoI, then inserts the multiple clone site of pGEX; Fusion protein expression vector pGEX-T4 PNK 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:
Respectively fusion protein expression vector pGEX-T4PNK and control vector pGEX are proceeded to expressive host bacterium Escherichiacoli BL21 (DE3), picking mono-clonal is seeded to the fresh LB substratum containing 50mg/l penbritin; Treat that bacterium grows to OD 600be 0.6 left and right, adding respectively final concentration is the expression of the IPTG induction T4PNK of 0.6mM; Be 15 ° by the Temperature Setting in Induction Process, induce and collect thalline after two hours.
Further, purified fusion protein step is specially following steps:
Collect the cell of one liter of fermented liquid, and resuspended with the PBS of 40ml ice bath, and PBS contains 137mM NaCl, 2.7mM KCl, 10mM Na 2hPO 412H 2o, 2mM KH 2pO 4, pH7.4, ice-bath ultrasonic lysing cell, centrifuging and taking supernatant, supernatant is by the GST resin of PBS pre-equilibration, and loading is complete fully washs with PBS, finally use GST elutriant, GST elutriant is that 0.154g GSH is dissolved in 50ml 50mM Tris-HCl, wash-out target protein; GST affinity chromatography has been removed most thalline foreign proteins, and the DNA of foreign protein and trace is further removed in ion-exchange.
Advantage of the present invention is: by vector construction by the gene clone of T4 polynucleotide kinase in pGEX carrier, under cold condition, with GST amalgamation and expression, expressing protein solubility is increased greatly.In addition, the purifying that makes T4 polynucleotide kinase that adds of GST label 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-T4 PNK;
Figure 1B is that the SDS-PAGE of restructuring T4 polynucleotide kinase protein expression analyzes;
Wherein, swimming lane 1 is albumen marker (KD), and swimming lane 2 and swimming lane 3 are expressed with the whole bacterial protein of the rear pGEX-T4PNK of induction before being respectively IPTG induction;
Fig. 2 is the optimization of T4 polynucleotide kinase expression condition;
Wherein, M: albumen marker (KD); T, S, I represent respectively whole protein, upper cleer and peaceful precipitation; 15 DEG C and 37 DEG C represent different inducing temperatures;
Fig. 3 is that the SDS-PAGE (12%) of the GST-T4 PNK after purifying analyzes:
Wherein, 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 washing; Swimming lane 5:buffer C washing; Swimming lane 6:buffer D wash-out fusion rotein; M: albumen marker (KD);
Fig. 4 is the determination of activity of the T4 polynucleotide kinase after purifying;
Embodiment
The present invention is by the structure of restructuring T4 polynucleotide kinase prokaryotic expression carrier pGEX-T4 PNK, success by codon optimized T4 polynucleotide kinase gene clone to the downstream of GST label coding sequence and with it in same reading frame, built the prokaryotic expression carrier of fusion rotein GST-T4 PNK, sequencing result shows that reading frame and DNA sequence dna are all correct.By contrast experiment, find that the expression amount of pGEX-T4 polynucleotide kinase albumen reaches 15% under low temperature induction condition, and solubility reaches 95%.GST affinity chromatography has been removed most thalline foreign proteins, and the DNA of foreign protein and trace is further removed in ion-exchange, has obtained the T4 polynucleotide kinase (purity approximately 95%) of about 7mg in one liter of fermented liquid.12%SDS-PAGE analyzes and shows, the T4 polynucleotide kinase molecular weight of purifying is 65kD.The T4 polynucleotide kinase of purifying shows and the similar activity of commercialization enzyme (NEB company), and specific activity is about 10000units/mg.
Below by preferred embodiment, the present invention is done to more detailed discussion.
The structure of restructuring T4 polynucleotide kinase prokaryotic expression carrier pGEX-T4 PNK:
Taking the T4 PNK that synthesizes as template, with the method amplification T4 PNK encoding sequence of PCR,
Forward primer used is: 5 '-CATG gGATCCaTGAAGAAAATCATT-3 ',
Reverse primer is: 5 '-CATG cTCGAGtCAAAAGTCCCCACTAG-3 '.
In forward primer, there is the restriction enzyme site (underscore) of a BamHI, in reverse primer, have the restriction enzyme site (underscore) of an XhoI.PCR reaction conditions is: 94 DEG C of thermally denatures 1 minute, and 55 DEG C of annealing 1min, 72 DEG C are extended 90 seconds, carry out altogether 25 circulations.The PCR product of purifying carries out double digestion with BamHI and XhoI, then inserts the multiple clone site of pGEX.Recombinant expression vector pGEX-T4 PNK 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 T4 PNK gene clone to the downstream of GST label coding sequence and with it in same reading frame.Recombinant plasmid, through sequence verification, has successfully built the prokaryotic expression carrier of fusion rotein T4 PNK, and reading frame and DNA sequence dna are all correct, and its sketch is referring to Figure 1A.
1, the expression of T4 PNK:
(1) expression of fusion rotein T4 PNK
Respectively T4 PNK fusion protein expression vector pGEX-T4 PNK and control vector pGEX are proceeded to expressive host bacterium Escherichiacoli BL21 (DE3), picking mono-clonal is seeded to fresh LB substratum (containing 50mg/l penbritin).Treat that bacterium grows to OD 600be 0.6 left and right, adding respectively final concentration is the expression of the IPTG induction T4 PNK of 0.6mM.Induce and collect thalline after two hours, tropina sample after treatment is walked 12%SDS-PAGE, use UVP white/ultraviolet transilluminator to carry out sweep record to the gel of coomassie brilliant blue staining, adopt specialty analysis software Grab-it2.5 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 65kD, and theoretical molecular size is identical, through gel image scanning gray analysis, T4 polynucleotide kinase fusion rotein accounts for 15% left and right (Figure 1B) of bacterial protein.(3) optimization of fusion rotein T4 PNK expression condition
Find through contrast experiment, under the inductive condition of 15 ° of low temperature, the solubility of fusion rotein T4 PNK increases, and is about 3 times under 37 DEG C of inductive conditions.Experimental result as shown in Figure 2.
(2) purifying of albumen
Collect the cell of one liter of fermented liquid, and with PBS (137mM NaCl, 2.7mM KCl, the 10mM Na of 40ml ice bath 2hPO 412H 2o, 2mM KH 2pO 4, pH7.4) and resuspended, ice-bath ultrasonic lysing cell, centrifuging and taking supernatant, supernatant is by the GST resin of PBS pre-equilibration, and loading is complete fully washs with PBS, finally uses GST elutriant (0.154g GSH is dissolved in 50ml50mM Tris-HCl pH8.0) wash-out target protein.GST affinity chromatography has been removed most thalline foreign proteins, and the DNA of foreign protein and trace is further removed in ion-exchange, has obtained the T4 polynucleotide kinase (purity approximately 95%) of about 7mg in one liter of fermented liquid.12%SDS-PAGE analyzes and shows, the T4 polynucleotide kinase molecular weight of purifying is 65kD (Fig. 3).
(4) activity determination method of T4 PNK
The activity of T4PNK adopt [γ- 32p] the ATP method of mixing carries out quantitative analysis, idiographic flow reference (Proc.Natl Acad.Sci.USA.54 (1965), 158-165).I.e. 1 Richardson unit of 1 unit, refers under 37 DEG C of conditions, in 30 minutes catalysis 1nmol acid insoluble [ 32p] mix needed enzyme amount.
As shown in Figure 4, in 50 μ lT4 polynueleotide kinase reaction buffer systems, 37 DEG C of incubations 30 minutes;?-phosphate group is transferred to 5 ' C-terminal of d (T) 8 from ATP, and [ATP and d (T) 8 ratios are 1: 1, amount is 50pmol], result shows that 20 unit enzyme amounts can make to exceed 90% phosphate group transfer.

Claims (5)

1. a recombinant T 4 bacteriophage polynucleotide kinase, is characterized in that the nucleotide sequence of described T4 phage polynucleotide kinase is as shown in sequence table SEQ ID NO:1.
2. the preparation method of a kind of recombinant T 4 bacteriophage polynucleotide kinase as claimed in claim 1, it is characterized in that, comprise the steps: by vector construction by codon optimized T4 phage polynucleotide kinase gene clone in pGEX carrier, under cold condition by with GST amalgamation and expression, improve expressing quantity and solubility; Described T4 phage polynucleotide kinase gene order is as shown in sequence table SEQ ID NO:1; T4 phage polynucleotide kinase and GST amalgamation and expression in Host Strains, amalgamation and expression albumen is GST-T4 PNK; Described amalgamation and expression albumen contains GST label, with GST pillar purified fusion protein.
3. the preparation method of a kind of recombinant T 4 bacteriophage polynucleotide kinase according to claim 2, is characterized in that described vector construction step is specially following steps:
Design of primers is as follows:
Forward primer used is: 5 '-CATG gGATCCaTGAAGAAAATCATT-3 '; Underscore part is the restriction enzyme site of BamHI;
Reverse primer used is: 5 '-CATG cTCGAGtCAAAAGTCCCCACTAG-3 '; Underscore part is the restriction enzyme site of XhoI;
Taking the T4 PNK that synthesizes as template, with the method amplification T4 PNK encoding sequence of PCR, PCR reaction conditions is: 94 DEG C of thermally denatures 1 minute, and 55 DEG C of annealing 1min, 72 DEG C are extended 90 seconds, carry out altogether 25 circulations; The PCR product of purifying carries out double digestion with BamHI and XhoI, then inserts the multiple clone site of pGEX; Fusion protein expression vector pGEX-T4 PNK is carried out to DNA sequencing, analyze the exactness of its reading frame and encoding sequence.
4. the preparation method of a kind of recombinant T 4 bacteriophage polynucleotide kinase according to claim 2, is characterized in that described amalgamation and expression step is specially following steps:
Respectively fusion protein expression vector pGEX-T4 PNK and control vector pGEX are proceeded to expressive host bacterium Escherichiacoli BL21 (DE3), picking mono-clonal is seeded to the fresh LB substratum containing 50mg/l penbritin; Treat that bacterium grows to OD 600be 0.6 left and right, adding respectively final concentration is the expression of the IPTG induction T4 PNK of 0.6mM; Be 15 ° by the Temperature Setting in Induction Process, induce and collect thalline after two hours.
5. the preparation method of a kind of recombinant T 4 bacteriophage polynucleotide kinase according to claim 2, 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 PBS of 40ml ice bath, and the formation of described PBS is 137mM NaCl, 2.7mM KCl, 10mM Na 2hPO 412H 2o, 2mM KH 2pO 4, pH7.4, ice-bath ultrasonic lysing cell, centrifuging and taking supernatant, supernatant is by the GST resin of PBS pre-equilibration, and loading is complete fully washs with PBS, finally use GST elutriant, the formation of described GST elutriant is that 0.154g GSH is dissolved in 50ml 50mM Tris-HCl, wash-out target protein; GST affinity chromatography has been removed most thalline foreign proteins, and the DNA of foreign protein and trace is further removed in ion-exchange.
CN201410361153.9A 2014-07-24 2014-07-24 Recombinant T4 bacteriophage polynucleotide kinase (T4 PNK) and preparation method thereof Pending CN104178467A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636029A (en) * 2016-12-12 2017-05-10 菲鹏生物股份有限公司 T4 polynucleotide kinase recombinase, preparation method thereof, expression gene, expression vector and host cell
WO2019193526A1 (en) * 2018-04-05 2019-10-10 Tsinghua University Methods of sequencing and producing nucleic acid sequences
CN114836448A (en) * 2022-05-19 2022-08-02 安诺优达基因科技(北京)有限公司 Nucleic acid molecule of codon-optimized T4 polynucleotide kinase and expression method thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636029A (en) * 2016-12-12 2017-05-10 菲鹏生物股份有限公司 T4 polynucleotide kinase recombinase, preparation method thereof, expression gene, expression vector and host cell
WO2018107521A1 (en) * 2016-12-12 2018-06-21 菲鹏生物股份有限公司 T4 polynucleotide kinase recombinase and preparation method, expression gene, expression vector, and host cell of same
WO2019193526A1 (en) * 2018-04-05 2019-10-10 Tsinghua University Methods of sequencing and producing nucleic acid sequences
CN114836448A (en) * 2022-05-19 2022-08-02 安诺优达基因科技(北京)有限公司 Nucleic acid molecule of codon-optimized T4 polynucleotide kinase and expression method thereof
CN114836448B (en) * 2022-05-19 2023-12-05 浙江安诺优达生物科技有限公司 Nucleic acid molecule of codon-optimized T4 polynucleotide kinase and expression method thereof

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