CN102653727A - Construction method and application of pyruvic acid-phosphoric acid double-kinase recombinant expression strain - Google Patents
Construction method and application of pyruvic acid-phosphoric acid double-kinase recombinant expression strain Download PDFInfo
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Abstract
The invention relates to a construction method of a pyruvic acid-phosphoric acid double-kinase recombinant expression strain, and specific application of the constructed strain, namely an expression and purification method of a pyruvic acid-phosphoric acid double-kinase, and application of the obtained pyruvic acid-phosphoric acid double-kinase in an ATP (adenosine triphosphate)-fluorescein-luciferase luminous reaction system. A pyruvic acid-phosphoric acid double-kinase coding gene ppdk from Streptomyces coelicolor is cloned and constructed into an expression vector pET28a-ppdk; the vector is transformed to an expression host BL21(DE3) to obtain a recombinant expression strain BL21(DE3)/pET28a-ppdk; the strain is cultured and induced to express the pyruvic acid-phosphoric acid double-kinase; and the pyruvic acid-phosphoric acid double-kinase is purified by affinity chromatography. The purified enzyme can effectively catalyze the reaction products adenosine monophosphate (AMP) and pyrophosphoric acid (PPi) to generate ATP in an ATP-fluorescein-luciferase luminous reaction system, and thus, has important application in ATP luminous detection.
Description
[technical field]
The present invention relates to mikrobe molecular biology and gene engineering technology field, relate to the clone of microprotein encoding sox, structure, the structure of recombinant strains, the two kinase whose expression of pyruvate phosphate, purifying and the application thereof of expression vector.
[background technology]
Two kinases (the Pyruvate Phosphate Dikinase of pyruvate phosphate; EC 2.7.9.1) the reversible catalysis phosphoric acid allyl formula pyruvic acid of ability, single adenosine phosphate (Adenosine Monophosphate; AMP) and pyrophosphate salt (Pyrophosphate; PPi) generate Triphosaden (Adenosine Triphosphate, ATP), inorganic phosphate (Orthophosphate) and pyruvic acid.Therefore, this enzyme can continue catalysis AMP and generate ATP in ATP-fluorescein-luciferase noclilucence system, and the mensuration that forms luminous signal in stable state noclilucence signal, the simplification testing process for system has positive effect.
In practical application, this enzyme can be used for the bacterial number in the ATP luminescence method rapid detection food.Zou Bingjie etc. have carried out researchs such as gene clone, expression to the two kinases of pyruvate phosphate in little pair of spore bacterium source of hot rose; And, be applied to catalytic chain reaction (Zou Bingjie, the Chen Ying of ATP-AMP with resulting enzyme and luciferase coupling; Ma Yinjiao; Zhou Guohua. two kinases of recombinant acetone acid phosphoric acid and luciferase coupling catalysis ATP-AMP circulating reaction [J]. Chinese biological chemistry and molecular biosciences journal, 2008,24 (11): 1081～1084.).
The present invention adopts sky blue chain enzyme bacteria (Streptomyces coelicolor) A3 (2) strain as starting strain; The two kinase genes of the coded pyruvate phosphate of this strain chromosome DNA are cloned; Recombinant expression vector and recombinant strains have been made up; And recombinant strains carried out abduction delivering; In ATP-fluorescein-luciferase noclilucence system, the activity of this enzyme is checked, the two kinases of pyruvate phosphate in sky blue chain enzyme bacteria A3 (2) strain of results suggest source have very high enzyme activity, and in the biloluminescence method of mikrobe detects, having very, important use is worth.
[summary of the invention]
[technical problem that will solve]
The objective of the invention is to make up a kind of two kinase whose recombinant strains of pyruvate phosphate that are suitable in sky blue chain enzyme bacteria A3 (2) strain of expression in escherichia coli source, and the two kinase whose methods of this recombinant strains abduction delivering pyruvate phosphate, the two kinase whose purification process of pyruvate phosphate and the applicating example of this enzyme are provided.Thereby the technical problem that the present invention will solve is exactly: make up two kinase expression carriers of pyruvate phosphate and required material and the method for recombinant strains; And two kinase whose abduction deliverings of pyruvate phosphate and required material and the method for purifying, and carry out required material and the method for practical application that ATP transforms.
[technical scheme]
The present invention adopts the two kinases of the pyruvate phosphate that derives from streptomyces coelicolor; Make up a kind of expression vector that this enzyme carries out abduction delivering that is fit to; Make up and be fit to the recombinant strains that this enzyme carries out abduction delivering; And the recombinant strains that adopts above-mentioned structure carries out the two kinase whose abduction deliverings of pyruvate phosphate, then the two kinases of this pyruvate phosphate carried out purifying and enzymic activity detection.
The host strain that the present invention adopts is e. coli bl21 (DE3); With the basic framework of commercially available commercialization coli expression carrier pET28a (+) as inducible expression carrier structure among the present invention; With streptomyces coelicolor A (3) 2 genomic dnas is template, through polymerase chain reaction (Polymerase Chain Reaction; Be called for short PCR) amplify the two kinase whose encoding sox ppdk of pyruvate phosphate, make up recombinant expression vector pET28a-ppdk, and, obtain recombinant strains BL21 (DE3)/pET28a-ppdk with this recombinant vectors transformed into escherichia coli BL21 (DE3).
Recombinant strains BL21 (DE3)/pET28a-ppdk cultivates in containing Luria-Bertani (LB) liquid nutrient medium of 50 μ g/mL kantlex; Adopt isopropyl-(IPTG) to carry out the two kinase whose abduction deliverings of pyruvate phosphate subsequently; Fermentation thalline behind the results abduction delivering; Carrying out ultrasonic disruption handles; The broken liquid of pair cell carries out centrifugal and results contain the two kinase whose supernatants of pyruvate phosphate, and expression of results shows through denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and analyzes.Adopt the affinity chromatography method that this enzyme is carried out purification process, the sample that purifying obtains shows through SDS-PAGE and analyzes.The two kinases of pyruvate phosphate that adopt ATP-fluorescein-luciferase noclilucence system that purifying is obtained carry out enzyme activity and detect and assess.
[embodiment]
(1) clone of the two kinases encoding soxs of pyruvate phosphate: be template at first with streptomyces coelicolor A3 (2) pnca gene group DNA; Adopt primer P1 and P2 (seeing table 1) to carry out pcr amplification and obtain the ppdk gene fragment, this fragment is carried out determined dna sequence to verify its exactness.Be used for next step experiment after order-checking is correct.
(2) structure of expression vector pET28a-ppdk: adopt restriction enzyme NdeI and XhoI that resulting ppdk gene fragment in the embodiment (1) is degraded; And reclaim this NdeI-XhoI fragment; Be connected to the corresponding restriction enzyme site (NdeI-XhoI) among the commercial intestinal bacteria inducible expression carrier pET28a (+), obtain recombinant expression plasmid called after pET28a-ppdk.
(3) structure of recombinant strains
With expression vector pET28a-ppdk transformed into escherichia coli BL21 (DE3); Obtain containing the intestinal bacteria recombinant bacterial strain of carrier pET28a-ppdk in the LB solid culture primary surface screening that contains 50 μ g/mL kantlex; With this bacterial strain called after BL21 (DE3)/pET28a-ppdk; This bacterial strain on January 4th, 2012 in China Committee for Culture Collection of Microorganisms common micro-organisms center (Beijing) preservation, its deposit number is CGMCC 5691.
(4) the two kinase whose abduction deliverings of pyruvate phosphate
E. coli bl21 (DE3)/pET28a-ppdk incubated overnight in the LB liquid nutrient medium is inoculated in 1% ratio and carries out shaking table in the LB liquid nutrient medium that contains kantlex and cultivate, as nutrient solution turbidity OD as seed liquor
600Reach at 0.6～0.8 o'clock, adopt IPTG to induce, carried out inducing culture 2～10 hours in 20 ℃～37 ℃, obtain fermented liquid, adopt supersonic method to abolish the somatic cells wall through centrifugal results thalline, and then centrifugal recovery supernatant.
(5) the two kinase whose separation and purification of pyruvate phosphate
Bacterium liquid behind the inducing culture is through centrifugal collection thalline, and with the resuspended thalline of binding buffer liquid, behind the employing ultrasonic disruption cell, centrifugal collection supernatant carries out affinitive layer purification.Adopt nickel chelating sepharose affinity media to fill chromatography column, recombinant protein is carried out purifying.In advance with level pad balance affinity column, with cleaning buffer solution wash-out foreign protein, use elution buffer wash-out recombinant protein again behind the sample upper prop before the upper prop, finally use the recombinant protein sample buffer.Elute protein is dialysed, purified recombinant protein is carried out SDS-PAGE analyze.
(6) the two kinase whose enzyme activity detection methods of pyruvate phosphate
The luciferase bioluminescence reaction changes into AMP with ATP; The two kinases of pyruvate phosphate can be converted into ATP with the AMP that produces; Circulating reaction like this; The ATP that continues to produce can convert the continual and steady strong luminescence signal of luciferin bioluminescence reaction to, detects this luminous signal and can estimate the two kinase whose activity of recombinant protein pyruvate phosphate.
Table 1 the present invention tests employed PCR primer
[beneficial effect of the present invention]
The present invention has following advantage: the two kinases stable in properties of the pyruvate phosphate in streptomyces coelicolor source have better heat-resisting property and chemical modifiability.This enzyme and luciferase carry out coupling and react, and can produce continual and steady fluorescent signal, for the bacterium in the samples such as biological luminescent method detection food provides method preferably.The present invention the C of the two kinase whose aminoacid sequences of recombinant acetone acid phosphoric acid hold into 6 histidine-tagged (His-tag) so that adopt the affinity chromatography method to carry out purifying.The catalytic ATP-AMP circulation of luciferase coupling can make enzyme recycle, and can significantly improve the sensitivity that ATP detects.
[description of drawings]
Fig. 1 recombinant expression vector pET28a-ppdk structural representation
The two kinases SDS-PAGE electrophoresis assays of the pyruvate phosphate that Fig. 2 purifying obtains.Swimming lane " M " is a molecular weight standard; Swimming lane " 1 " is the two kinases electrophoretic bands of the pyruvate phosphate of purifying
The two kinases of Fig. 3 pyruvate phosphate are to the effect of the fluorescence radiation buffer system that do not contain ATP.The luminescence kinetics curve of A system when adding the two kinases of pyruvate phosphate wherein; The luminescence kinetics curve of B system when not adding the two kinases of pyruvate phosphate
The two kinases of Fig. 4 pyruvate phosphate are to the effect of the fluorescence radiation buffer system that contains ATP.The luminescence kinetics curve of A system when adding the two kinases of pyruvate phosphate wherein; The luminescence kinetics curve of B system when not adding the two kinases of pyruvate phosphate
[embodiment]
Describe process of the present invention in detail below in conjunction with accompanying drawing.
The structure of two kinases recombinant expression vectors of embodiment 1 pyruvate phosphate and recombinant strains
(1) clone of the two kinases encoding soxs of pyruvate phosphate
The two kinases encoding soxs of the pyruvate phosphate that the present invention adopts come from streptomyces coelicolor A3 (2) strain, obtain target fragment through the PCR reaction from streptomyces coelicolor A3 (2) pnca gene group DNA amplification.
Be template at first, adopt primer P1 and P2 (seeing table 1) to carry out pcr amplification gene ppdk with streptomyces coelicolor A3 (2) pnca gene group DNA.Wherein, 5 ' of primer P1-end is introduced restriction endonuclease sites (hereinafter to be referred as restriction enzyme site) NdeI, and 3 ' of primer P2-end is introduced restriction enzyme site XhoI.
The actual conditions of PCR reaction is: 95 ℃ of preparatory sex change 5min, and 95 ℃ of sex change 45s, 68 ℃ of renaturation 45s, 72 ℃ are extended 2min50s, and 30 circulations are carried out in reaction, and system temperature is reduced to 4 ℃ of extension 10min then.
The product that the PCR reaction is obtained carries out the agarose gel electrophoresis analysis, and the length that this fragment that pcr amplification goes out shows on 1% agarose gel electrophoresis is about 2700 base pairs, and (1 base pair is abbreviated as 1bp, and 1000 base pairs are abbreviated as 1kb; This fragment is reclaimed and serves Hai Boshang Bioisystech Co., Ltd and carry out determined dna sequence to verify its exactness down together).
(2) structure of recombinant expression vector pET28a-ppdk and expression strain BL21 (DE3)/pET28a-ppdk
The ppdk gene fragment that above-mentioned sequence verification sequence encoding is correct is degraded with restriction enzyme NdeI and XhoI; Reclaim the endonuclease bamhi about 2.7kb; And this fragment cloning obtained recombinant secretor expression vector pET28a-ppdk in escherichia coli vector pET28a (+), the structural representation of this carrier is as shown in Figure 1.Adopt Calcium Chloride Method to prepare the competent cell of e. coli bl21 (DE3); And expression vector pET28a-ppdk is transformed in the above-mentioned competent cell; Then conversion fluid is coated on the LB solid culture primary surface that contains 50 μ g/mL kantlex; In 37 ℃ of cultivation 8～12h, obtain containing the intestinal bacteria recombinant bacterial strain of carrier pET28a-ppdk then from this solid culture primary surface screening, these recombinant bacterial strains are carried out the extracting of plasmid vector; And adopt NdeI-XhoI double digestion checking institute extracting plasmid size whether to conform to the molecular size of expression vector pET28a-ppdk, verify correct bacterial strain called after BL21 (DE3)/pET28a-ppdk.
Two kinase whose abduction deliverings of embodiment 2 pyruvate phosphates and separation and purification
With e. coli bl21 (DE3)/pET28a-ppdk in the LB liquid nutrient medium incubated overnight as seed liquor; Be inoculated in the LB liquid nutrient medium that contains 50 μ g/mL (ultimate density) kantlex in 1% ratio; In 37 ℃, the cultivation of 200r/min shaking table, as nutrient solution turbidity OD
600Reach at 0.6～0.8 o'clock; The employing final concentration is that the IPTG of 0.05～1.0mmol/L induces; Carried out inducing culture 2～10 hours in 20 ℃～37 ℃, obtain fermented liquid and under the condition of 0 ℃～6 ℃ of 8000～12000r/min and temperature, carry out centrifugal 8～12min results thalline.The thalline of results is resuspended in binding buffer liquid PBS (prescription: 50mmol/L Tris-HCl, 500mmol/L NaCl, 20～100mmol/L imidazoles, pH 7.4), adopts supersonic method (temperature: 0～6 ℃ then; Ultrasonic power: 200～800W; UW working hour 1s, quiescent interval 9s, the omnidistance working hour 30～60min) is abolished the somatic cells wall; And then under the condition of 0～6 ℃ of 8000～12000r/min and temperature, carry out centrifugal 8～12min, and reclaim supernatant.
Adopt nickel chelating sepharose affinity media to fill chromatography column the two kinases of recombinant acetone acid phosphoric acid that reclaim in the supernatant are carried out purifying.Following purge process temperature of living in is 4 ℃.Before the upper prop in advance with level pad (50mmol/L Tris-HCl, 500mmol/LNaCl, 20～100mmol/L imidazoles of 5～10 times of column volumes; PH 7.4) the balance affinity column, behind the sample upper prop with cleaning buffer solution (50mmol/LTris-HCl, 500mmol/L NaCl; 50～80mmol/L imidazoles, pH 7.4) the wash-out foreign protein, use elution buffer (50mmol/L Tris-HCl again; 500mmol/L NaCl; 200～500mmol/L imidazoles, pH 7.4) the wash-out recombinant protein, the enzyme appearance to wash-out is carried out dialysis treatment then.
The SDS-PAGE of the two kinase expression products of embodiment 3 recombinant acetone acid phosphoric acids detects
The two kinases samples of embodiment 2 described purified pyruvate phosphates are carried out SDS-PAGE (12% polyacrylamide gel) electrophoretic analysis, and the result is as shown in Figure 2.It is thus clear that near the top of 97.4kDa band comparatively single sample strip (swimming lane is designated as " 1 ") is arranged at molecular weight standard (swimming lane is designated as " M "); The two monomeric theoretical moleculars of kinases of pyruvate phosphate are approximately 97.6kDa, so experimental result is consistent with the theoretical molecular size.
The two kinases applicating examples () of embodiment 4 pyruvate phosphates
Reaction and luciferase effect that the two kinase catalytic AMP of pyruvate phosphate generate ATP make ATP generate the reaction generation coupling of AMP; The two kinases of pyruvate phosphate are converted into ATP with AMP; Luciferase changes into AMP with ATP again, circulating reaction like this, and the ATP that continues to produce can make resorcinolphthalein send continual and steady luminous signal; For changing ATP-fluorescein-luciferase bioluminescence reaction kinetics, realize that the detection of luminous signal is oversimplified significant.
Preparation does not contain the fluorescein-luciferase reaction system of ATP: totally 400 μ L, and comprising AMP (final concentration 1 * 10
-6Mol/L), PPi (final concentration 1 * 10
-6Mol/L), resorcinolphthalein (final concentration 5 * 10
-6Mol/L), luciferase (final concentration 3 * 10
-7Mol/L), Tris-HCl 25mmol/L, system pH 7.4.
Wherein add the two kinases 100 μ L of pyruvate phosphate that purifying obtains described in the embodiment 2～3 to this reaction system, utilize Chemiluminescence Apparatus to detect the fluorescent signal that is generated by ATP then, its result is shown in curve A among Fig. 3; As empty map, when not adding the two kinases of purifying obtains described in the embodiment 2～3 pyruvate phosphate, fluorescent signal is shown in curve B among Fig. 3.This result shows that the two kinases of the pyruvate phosphate that purifying obtains described in the embodiment 2～3 can promote the generation of ATP in this system and the increase of ATP concentration, and in limitting detection time, can realize the enhancing of fluorescent signal level.
The two kinases applicating examples (two) of embodiment 5 pyruvate phosphates
Experimentize according to embodiment 4 described modes, just in reaction system, remove AMP and PPi, (final concentration is 1 * 10 to add ATP
-6Mol/L), other components unchanged.
Wherein add the two kinases 100 μ L of pyruvate phosphate that purifying obtains described in the embodiment 2～3 to this reaction system, utilize Chemiluminescence Apparatus to detect the fluorescent signal that is generated by ATP then, its result is shown in curve A among Fig. 4; As empty map, when not adding the two kinases of purifying obtains described in the embodiment 2～3 pyruvate phosphate, fluorescent signal is shown in curve B among Fig. 4.This result shows, the two kinases of the pyruvate phosphate that purifying obtains described in the embodiment 2～3 can promote keeping of ATP concentration in this system, and in the detection time limit, can realize keeping of fluorescent signal level.
Claims (5)
1. express the construction process and the application thereof of producing two kinase whose recombination bacillus coli (Escherichia coli) the bacterial strain BL21 (DE3) of pyruvate phosphate/pET28a-ppdk for one kind.This bacterial strain on January 4th, 2012 in China Committee for Culture Collection of Microorganisms common micro-organisms center (Beijing) preservation, its deposit number is CGMCC 5691.
2. coli strain BL21 according to claim 1 (DE3)/pET28a-ppdk and application thereof is characterized in that the construction process of this bacterial strain is following:
(1) clone of the two kinases encoding soxs of pyruvate phosphate
DNA is a template with streptomyces coelicolor (Streptomyces coelicolor) A3 (2) pnca gene group; Adopt primer P1 and P2 to carry out polymerase chain reaction (PCR); Amplification obtains the two kinase whose encoding sox ppdk (Gene ID:1095632) of pyruvate phosphate; This gene is about 2.7kb, has 6 histidine-tagged (His-Tag).
(2) structure of expression vector and recombinant bacterial strain
Ppdk degrades with restriction enzyme NdeI and XhoI with this gene; Reclaim the NdeI-XhoI fragment and be cloned into the NdeI-XhoI site among the expression vector pET28a (+); Obtain recombinant expression plasmid pET28a-ppdk; With this carrier transformed into escherichia coli bacterial strain BL21 (DE3), obtain recombinant bacterial strain coli strain BL21 (DE3)/pET28a-ppdk.
3. coli strain BL21 according to claim 1 (DE3)/pET28a-ppdk and application thereof is characterized in that two kinase whose abduction deliverings of pyruvate phosphate and separation purification method are following:
Coli strain BL21 (DE3)/pET28a-ppdk is cultivated in the Luria-Bertani medium liquid that has added kantlex (final concentration is 50 μ g/mL) (being called for short the LB substratum); In the time of between nutrient solution turbidity OD600 reaches 0.6～0.8; Adopt final concentration be the isopropyl-(IPTG) of 0.05～1.0mmol/L as inductor, between 20 ℃～37 ℃, carried out inducing culture 2～10 hours.
4. coli strain BL21 according to claim 1 (DE3)/pET28a-ppdk and application thereof is characterized in that the separation purification method of the two kinases abduction delivering products of pyruvate phosphate is following:
Reclaim the fermented liquid thalline and adopt UW to abolish cell walls, the supernatant that obtains is filled chromatography column through nickel chelating sepharose affinity media, the two kinases of the pyruvate phosphate of reorganization are carried out separation and purification.
5. coli strain BL21 according to claim 1 (DE3)/pET28a-ppdk and application thereof, the primer sequence of the two kinases encoding sox ppdk of pyruvate phosphate that it is characterized in that increasing is following:
-P1.5′-GTCTATCATATGGCCCGTTACGTGTACGAC-3′
P2：5′-TATATACTCGAGTCGGCTGTCGCCGGCATCG-3′?。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110779887A (en) * | 2019-09-30 | 2020-02-11 | 浙江大学 | Method for determining phosphoglycerate kinase activity |
| CN110869510A (en) * | 2017-07-12 | 2020-03-06 | 埃科莱布美国股份有限公司 | Rapid method for detecting bacterial spores |
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| CN1135770A (en) * | 1994-07-29 | 1996-11-13 | 日本烟草产业株式会社 | Polypeptide having cold-resistant pyruvate phosphate dikinase activity, DNA coding for the polypeptide, recombinant vector containing the DNA and transformed plant |
| WO2010097623A1 (en) * | 2009-02-27 | 2010-09-02 | Advanced Technologies (Cambridge) Limited | Transgenic plants comprising constructs encoding phosphoenolpyruvate carboxykinase and/or pyruvate ortho phos phate dikinase |
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| CN1135770A (en) * | 1994-07-29 | 1996-11-13 | 日本烟草产业株式会社 | Polypeptide having cold-resistant pyruvate phosphate dikinase activity, DNA coding for the polypeptide, recombinant vector containing the DNA and transformed plant |
| WO2010097623A1 (en) * | 2009-02-27 | 2010-09-02 | Advanced Technologies (Cambridge) Limited | Transgenic plants comprising constructs encoding phosphoenolpyruvate carboxykinase and/or pyruvate ortho phos phate dikinase |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110869510A (en) * | 2017-07-12 | 2020-03-06 | 埃科莱布美国股份有限公司 | Rapid method for detecting bacterial spores |
| CN110779887A (en) * | 2019-09-30 | 2020-02-11 | 浙江大学 | Method for determining phosphoglycerate kinase activity |
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Application publication date: 20120905 |