CN103275995A - Firefly luciferase gene and application thereof - Google Patents
Firefly luciferase gene and application thereof Download PDFInfo
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- CN103275995A CN103275995A CN2013101724561A CN201310172456A CN103275995A CN 103275995 A CN103275995 A CN 103275995A CN 2013101724561 A CN2013101724561 A CN 2013101724561A CN 201310172456 A CN201310172456 A CN 201310172456A CN 103275995 A CN103275995 A CN 103275995A
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- firefly luciferase
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- 108090000331 Firefly luciferases Proteins 0.000 title claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000004472 Lysine Substances 0.000 claims abstract description 4
- 241000254064 Photinus pyralis Species 0.000 claims description 14
- 150000001413 amino acids Chemical group 0.000 claims description 3
- 230000035772 mutation Effects 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 11
- 238000000034 method Methods 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 3
- 108020004705 Codon Proteins 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 108010006591 Apoenzymes Proteins 0.000 abstract 1
- 235000004279 alanine Nutrition 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 abstract 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108060001084 Luciferase Proteins 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 4
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a firefly luciferase gene and an application thereof. The firefly luciferase gene is a mutLuc gene which is obtained through mutating the 31st-bit lysine of a wild type firefly luciferase gene into alanine and is used for producing firefly luciferase. Compared with a convention firefly luciferase production method, the wild type firefly luciferase gene is mutated and a Luc codon is optimized, so that the mutated firefly luciferase has the characteristics of easiness in purification, high yield (each litre of zymocyte can obtain more than 5mg of apoenzyme), high enzymic purity (enzymic specific activity is not less than 1*10<11> remaining useful life (RUL)/mg protein), high activity, strong stability and the like; and the method can efficiently produce the firefly luciferase, and is suitable for industrial production and applied to individual diagnosis.
Description
Technical field
The present invention relates to a kind of new firefly luciferase gene, the particularly application of this gene in producing Photinus pyralis LUC.
Background technology
Luciferase is the general designation that occurring in nature can produce noctilcent enzyme, and wherein most representative is that a kind of formal name used at school is the interior luciferase of light of firefly polypide of Phot inus pyralis.In the corresponding chemical reaction, the generation of fluorescence is the oxidation that comes from luciferin, also comprises Triphosaden (ATP) in some cases in the reaction system.In the absence of luciferase, the speed of luciferin and oxygen reaction is very slow, and usually further accelerated reaction of the existence of calcium ion (similar to the situation of Muscle contraction).Chemical reaction is as follows:
Luciferin+ATP+O2 → oxygen luciferin+PPi+AMP+CO2+ light.Energy is saved in this reaction very much, and the energy of nearly all input reaction all is converted into light.Under the situation that has excessive substrate and enzyme to exist, emitted luminescence intensity is directly proportional with ATP in the reaction system, therefore this characteristics of luminescence make Photinus pyralis LUC can and produce in the research of various mensuration ATP in widespread use, as the drug sensitive test of tumour individuation medical treatment.
The Photinus pyralis LUC of using mainly is that the wild-type Photinus pyralis LUC is cloned in the recombinant vectors at present, forwards expression in escherichia coli and purifying to.Jeffrey1985 has just made up the recombinant vectors pkw101 that contains firefly luciferase gene, the system production that utilizes Jeffrey to make up, need be through 30 minutes processing of 45 degree thermal inductions, had a strong impact on enzymic activity, there is separation difficulty equally in other people production method, serious problems such as yield is low, and enzyme work is low.
Summary of the invention
Defective at present prior art existence, the invention discloses a kind of firefly luciferase gene and application thereof, make product be easy to purifying, the output height, characteristics such as activity is high, and stability is strong are a kind of methods of producing Photinus pyralis LUC efficiently, be fit to industrial production, use in the individuation diagnosis.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
A kind of firefly luciferase gene is to be the mutLuc gene that obtains behind the L-Ala by the 31st lysine mutation with the firefly luciferase gene of wild-type.
Further, the nucleotide sequence of the firefly luciferase gene of described wild-type such as SEQ.ID.NO.1, the aminoacid sequence of described L-Ala such as SEQ.ID.NO.2.
A kind of firefly luciferase gene is for the production of the application in the Photinus pyralis LUC.
In described Pet-mutLuc, because the Photinus pyralis LUC sudden change is under the control of efficient promoter T7 gene and lactose operon Lac, when existing, firefly luciferase gene of the present invention can be by efficient inducible transcription at certain density sec.-propyl-b-D-thiogalactoside (IPTG).
Utilize pack into Xho1I and the BamHI of recombinant expression vector pet-15b of described mutLuc gene to obtain Pet-mutLuc, and recombinant vectors changed in the host bacterium (e. coli bl21 DE3), purifying obtains luciferase after described sec.-propyl-b-D-thiogalactoside (IPTG) is induced.
Compared with prior art, the present invention has the following advantages:
Compare with existing luciferase production method, the present invention is because the codon that has suddenlyd change the wild-type firefly luciferase gene and optimized Luc, make the Photinus pyralis LUC of this sudden change have the purifying of being easy to, yield height (every liter of zymophyte obtains zymoprotein greater than 5mg), enzyme purity height (specific activity of enzyme 〉=1 * 1011RUL/mg albumen), characteristics such as active high and stability is strong are a kind of methods of High-efficient Production Photinus pyralis LUC, be fit to industrial production, use in the individuation diagnosis.
Above-mentioned explanation only is the general introduction of technical solution of the present invention, for can clearer understanding technique means of the present invention, and can be implemented according to the content of specification sheets, below with preferred embodiment of the present invention and conjunction with figs. describe in detail as after.
Description of drawings
Accompanying drawing described herein is used to provide further understanding of the present invention, constitutes the application's a part, and illustrative examples of the present invention and explanation thereof are used for explaining the present invention, do not constitute improper restriction of the present invention.In the accompanying drawings:
Fig. 1 is recombinant expression vector Pet-mutLuc structural representation of the present invention;
Fig. 2 is for changeing the thalli growth graphic representation of bacterial strain under the IPTG inductive condition that Pet-mutLuc is arranged;
Fig. 3 induces different time enzyme activity determination graphic representation for IPTG;
Fig. 4 is the enzyme activity determination figure as a result of Photinus pyralis LUC in the A1-A10 collection tube.
Embodiment
Below with reference to the accompanying drawings and in conjunction with the embodiments, describe the present invention in detail.
A kind of firefly luciferase gene is to be the mutLuc gene that obtains behind the L-Ala by the 31st lysine mutation with the firefly luciferase gene of wild-type.
Further, the nucleotide sequence of the firefly luciferase gene of described wild-type such as SEQ.ID.NO.1, the aminoacid sequence of described L-Ala such as SEQ.ID.NO.2.
A kind of firefly luciferase gene is for the production of the application in the Photinus pyralis LUC.
Referring to shown in Figure 1, in described Pet-mutLuc, because the Photinus pyralis LUC sudden change is under the control of efficient promoter T7 gene and lactose operon Lac, when existing, firefly luciferase gene of the present invention can be by efficient inducible transcription at certain density sec.-propyl-b-D-thiogalactoside (IPTG).
Utilize pack into the Xho1 I of recombinant expression vector pet-15b of described mutLuc gene to draw BamHI and obtain Pet-mutLuc, and recombinant vectors changed in the host bacterium (e. coli bl21 DE3), purifying obtains luciferase after described sec.-propyl-b-D-thiogalactoside (IPTG) is induced.
Wild-type Pet-Luc and Pet-mutLuc are transformed into respectively in the competent cell of e. coli bl21 (DE3), the inoculation above-mentioned bacterial strains is in the LB substratum of 10ml, 37 degree shaken overnight, the 10ml bacterium that spends the night is inoculated in the fresh LB substratum of 1L, 37 degree continue to cultivate, be about 0.6 up to OD600, the adding final concentration is that the IPTG of 3mM induces different time.
Referring to shown in Figure 2, after IPTG induces, respectively 1,2, waited the different time sampling in 3,4,5,6 hours, utilize spectrophotometric determination OD600, obtain cell concentration.
Referring to shown in Figure 3, collect and induce the bacterium liquid 1ml of different time points, detect enzymic activity after the centrifugal cracking, the result shows that the enzymic activity of Pet-mutLuc group bacterium crude extract is apparently higher than wild-type.
Referring to shown in Figure 4, further behind the tweezer column purification, to get the 1ul purified product respectively and do the uciferase activity detection, result's demonstration is significantly higher than wild-type from the protease activity of Pet-mutLuc group purifying.
The above is the preferred embodiments of the present invention only, is not limited to the present invention, and for a person skilled in the art, the present invention can have various changes and variation.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Sequence table
Claims (3)
1. firefly luciferase gene is characterized in that: be to be the mutLuc gene that obtains behind the L-Ala by the 31st lysine mutation with the firefly luciferase gene of wild-type.
2. firefly luciferase gene according to claim 1 is characterized in that: the nucleotide sequence of the firefly luciferase gene of described wild-type such as SEQ.ID.NO.1, the aminoacid sequence of described L-Ala such as SEQ.ID.NO.2.
3. a firefly luciferase gene is for the production of the application in the Photinus pyralis LUC.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310172456.1A CN103275995B (en) | 2013-05-10 | 2013-05-10 | Firefly luciferase gene and application thereof |
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| CN201310172456.1A CN103275995B (en) | 2013-05-10 | 2013-05-10 | Firefly luciferase gene and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114350627A (en) * | 2021-12-16 | 2022-04-15 | 大连博格林生物科技有限公司 | Firefly luciferase mutant and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102597231A (en) * | 2009-11-10 | 2012-07-18 | 龟甲万株式会社 | Firefly luciferase and gene thereof, and process for production of firefly luciferase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102597231A (en) * | 2009-11-10 | 2012-07-18 | 龟甲万株式会社 | Firefly luciferase and gene thereof, and process for production of firefly luciferase |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114350627A (en) * | 2021-12-16 | 2022-04-15 | 大连博格林生物科技有限公司 | Firefly luciferase mutant and preparation method thereof |
| CN114350627B (en) * | 2021-12-16 | 2022-08-05 | 大连博格林生物科技有限公司 | Firefly luciferase mutant and preparation method thereof |
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