CN102492697A - Method for cloning and expressing nitrilre hydratase regulatory protein - Google Patents
Method for cloning and expressing nitrilre hydratase regulatory protein Download PDFInfo
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- CN102492697A CN102492697A CN2011103840892A CN201110384089A CN102492697A CN 102492697 A CN102492697 A CN 102492697A CN 2011103840892 A CN2011103840892 A CN 2011103840892A CN 201110384089 A CN201110384089 A CN 201110384089A CN 102492697 A CN102492697 A CN 102492697A
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- nitrile hydratase
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Abstract
The method discloses a method for cloning and expressing nitrilre hydratase regulatory protein, which is characterized in that His-tag is added in the N end of a gene sequence of the nitrilre hydratase regulatory protein and is serially connected with a nitrilre hydratase maturase gene, and the His-tag and the nitrilre hydratase maturase gene are co-expressed in escherichia coli Bl21. The invention belongs to the technical field of gene cloning. The method is simple in operation, and has higher efficiency and success rate.
Description
Technical field:
A kind of method of clonal expression Nitrile hydratase modulin is specifically related to a kind of method of in e. coli bl21, expressing the Nitrile hydratase modulin.
Background technology:
Nitrile hydratase (Nitrile hydratase is called for short NHase, EC 4.2.1.84) is the metalloenzyme that a kind of catalysis itrile group compound changes amido compounds into.Nearly 1,000,000 tons of the acrylic amide of producing with this kind of enzyme accounts for 1/3rd of whole acrylic amide output.Biotechnology with respect to traditional chemical method have that cost is low, the advantage of less energy consumption, less contamination.At present, in developed countries such as the U.S., Japan, France, this biotechnology is replacing traditional chemical method.In China, though the biotechnology of Nitrile hydratase synthesis of acrylamide start late, develop very fast, 2008 annual datas statistics, the acrylic amide of China's biological process production accounts for 41% of whole acrylic amide output.Acrylic amide is a kind of widely used basic chemical industry raw material, plays an important role at aspects such as oil production, papermaking, decorations.Along with the continuous expansion in market, to the demand of acrylic amide also in continuous growth.This kind of enzyme also is used in the production of nicotinamide in addition.Nicotinamide is a kind of B group VITAMINs, participates in organism metabolism with nicotinamide adenine dinucleotide and coenzyme II form, playing crucial effect aspect human body and the animal metabolism, is widely used on medicine, foodstuff additive and the fodder prodn.
The widespread use of Nitrile hydratase impels scientific worker the begin one's study synthesis mechanism and the catalytic mechanism of this metalloenzyme.Nitrile hydratase is divided into iron content and contains two kinds of cobalts by the difference of institute's metal ion, can be divided into four types again by the difference of gene structure.Though they are catalytic to be same type of chemical reaction, these a few zymoid synthesis models are different, and these different outstanding behaviours are in the difference of metals ion picked-up mode.Wherein the picked-up mechanism of cobalt ion is distinguished in the first kind and the second type of Nitrile hydratase, and the 3rd type of report not also, the related conclusions of the 4th type of Nitrile hydratase (containing swage) is also indeterminate.The key reason that obstruction is verified the 3rd type of cobalt ion picked-up mechanism is exactly that such Nitrile hydratase (is representative with the pseudomonas putida Nitrile hydratase) modulin does not still have the method for successful clonal expression at present.Its reason is: at present in the pseudomonas putida ORF of Nitrile hydratase regulatory gene sequence partly report wrong, even and correctly discern the ORF of this modulin, this modulin also is difficult to detect on SDS-PAGE.
Summary of the invention:
The invention provides a kind of ORFs of discerning the Nitrile hydratase modulin, its nucleotides sequence is classified SEQ ID NO.1 as.
The present invention also provides a kind of method of using above-mentioned ORFs clonal expression Nitrile hydratase modulin.
For addressing the above problem, provide technical scheme following:
1) downloading GenBank number from NCBI is sequence and the identification SEQ ID NO.1 of U89363.1;
2) before the regulatory protein gene sequence, add SD sequence (AAGGAG) and His-tag sequence;
3) will go up the sequence that makes up in the step and be connected on Nitrile hydratase maturing enzyme downstream and be cloned into pET-24a (+), import the e. coli bl21 abduction delivering;
4) the Nitrile hydratase enzyme was lived after HPLC detected and transforms.
Nitrile hydratase gene according to the invention is to derive from pseudomonas putida (Pseudomonas putida) NRRL-18668 (" A stereoselective cobalt-containing nitrile hydratase " is published in 1997 Biochemistry).
Detailed steps of the present invention is:
(1) the regulatory protein gene sequence that acquisition is correct is also discerned correct ORF
Through consulting correct respectively amplification maturing enzyme gene order of Nitrile hydratase gene order design primer and regulatory protein gene sequence in the pseudomonas putida.Because the ORF of Nitrile hydratase regulatory gene sequence partly reports wrong (this regulatory protein gene sequence has 3 kinds of possible ORF) in the pseudomonas putida at present; And this modulin is difficult to detect at SDS-PAGE, so increased difficulty for the ORF of correct identification modulin.Through attempting various ORF and adding the His-tag protection at this modulin N end, successful expression is gone out to have the proteic ORF of activity regulation, finally confirm as correct modulin ORF;
(2) regulatory protein gene sequence N end adds His-tag
Express natural Nitrile hydratase complete sequence in the pseudomonas putida in the e. coli bl21, in the world have its modulin of report can not successful expression more, through adding His-tag at regulatory protein gene sequence N end, but the successful expression modulin;
(3) overlapping PCR series connection Nitrile hydratase maturing enzyme and regulatory protein gene sequence
Through overlapping PCR the regulatory protein gene sequence that Nitrile hydratase N end is added with His-tag is connected on maturing enzyme gene order downstream;
(4) being cloned into pET-24a (+) imports in the e. coli bl21 and expresses
The Nitrile hydratase complete sequence that makes up in the last step is cloned into pET-24a (+) imports abduction delivering in the e. coli bl21;
(5) the Nitrile hydratase enzyme was lived after HPLC detected and transforms
Nitrile hydratase HPLC testing conditions: moving phase phosphoric acid acetonitrile damping fluid; Detect wavelength 210nm; Chromatographic column adopting C18 post;
(6) Nitrile hydratase modulin N end order-checking
The albumen of expressing is crossed N end order-checking behind the ni-sepharose purification; Sequencing result (seeing accompanying drawing 3) is the corresponding sequence of His-tag; And this His-tag artificial interpolation that be us; This just explains that this albumen is exactly the Nitrile hydratase modulin, lays a good foundation with the industrialized producing technology that improves Nitrile hydratase for further resolving Nitrile hydratase metals ion picked-up mechanism.
Beneficial effect of the present invention: the invention provides a kind of method of clonal expression Nitrile hydratase modulin, this method can be in e. coli bl21 successful expression Nitrile hydratase modulin.
Description of drawings:
Fig. 1. the full gene PCR of Nitrile hydratase
1, molecular weight of albumen standard; 2, the full gene PCR of Nitrile hydratase (regulatory protein gene N end adds His-tag).
Fig. 2. the SDS-PAGE electrophorogram that Nitrile hydratase is expressed
1, molecular weight of albumen standard; 2, (contain pET-24a (+) and Nitrile hydratase gene complete sequence wherein modulin N end add His-tag) full cell.
Fig. 3. N end sequencer map behind the Nitrile hydratase modulin purifying
Embodiment:
Material and detection method
Bacterial classification is pseudomonas putida (Pseudomonasputida) NRRL-18668, and pertinent literature " A stereoselective cobalt-containing nitrile hydratase " is published on 1997 the Biochemistry.
Nitrile hydratase HPLC testing conditions: moving phase phosphoric acid acetonitrile damping fluid; Detect wavelength 210nm; Chromatographic column is the C18 post.
1) the regulatory protein gene sequence that acquisition is correct is also discerned correct ORF
Through consulting correct respectively amplification maturing enzyme gene order of Nitrile hydratase gene order design primer and regulatory protein gene sequence in the pseudomonas putida.Because the ORF of Nitrile hydratase regulatory gene sequence partly reports wrong (this regulatory protein gene sequence has 3 kinds of possible ORF) in the pseudomonas putida at present; And this modulin is difficult to detect at SDS-PAGE, so increased difficulty for the ORF of correct identification modulin.Through attempting various ORF and adding the His-tag protection at this modulin N end, successful expression is gone out to have the proteic ORF of activity regulation, finally confirm as correct modulin ORF;
2) regulatory protein gene sequence N end adds His-tag
Express natural Nitrile hydratase complete sequence in the pseudomonas putida in the e. coli bl21, in the world have the report modulin can not successful expression more, through adding His-tag at regulatory protein gene sequence N end, but the successful expression modulin;
3) overlapping PCR series connection Nitrile hydratase maturing enzyme and regulatory protein gene sequence
Through overlapping PCR the regulatory protein gene sequence that Nitrile hydratase N end is added with His-tag is connected on maturing enzyme gene order downstream;
4) being cloned into pET-24a (+) imports in the e. coli bl21 and expresses
Nitrile hydratase complete sequence in the last step is cloned into pET-24a (+) imports abduction delivering in the e. coli bl21;
5) the Nitrile hydratase enzyme was lived after HPLC detected and transforms
Nitrile hydratase HPLC testing conditions: moving phase phosphoric acid acetonitrile damping fluid; Detect wavelength 210nm; Chromatographic column adopting C18 post;
6) Nitrile hydratase modulin N end order-checking
The albumen of expressing is crossed N end order-checking behind the ni-sepharose purification; Sequencing result (seeing accompanying drawing 3) is the corresponding sequence of His-tag; And this His-tag artificial interpolation that be us; This just explains that this albumen is exactly the Nitrile hydratase modulin, lays a good foundation with the industrialized producing technology that improves Nitrile hydratase for further resolving Nitrile hydratase metals ion picked-up mechanism.
Claims (6)
1. an ORFs of discerning the Nitrile hydratase modulin is characterized in that, its nucleotides sequence is classified SEQ ID NO.1 as.
2. the method for a clonal expression Nitrile hydratase modulin is to add SD sequence and His-tag at the said ORFs front end of claim 1, connects coexpression in e. coli bl21 with Nitrile hydratase maturing enzyme gene.
3. method as claimed in claim 2 is characterized in that, said Nitrile hydratase gene order derives from pseudomonas putida (Pseudomonas putida) NRRL-18668.
4. method as claimed in claim 2 is characterized in that, makes up pUC19-AB (his-tag) P cloning vector and pET-24a (+)-AB (his-tag) P expression vector.
5. method as claimed in claim 2 is characterized in that, said expressive host bacterium is an e. coli bl21.
6. like the described arbitrary method of claim 2-5, it is characterized in that the recombination bacillus coli fermention medium of said expression Nitrile hydratase modulin is: 1.2% Tryptones, 2.4% yeast extract, 0.4% glycerine, 17mMKH
2PO4,72mMK
2HPO4.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020147031A1 (en) * | 2019-01-16 | 2020-07-23 | 江南大学 | Nitrile hydratase mutant, genetically engineered bacterium containing same, and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010055666A1 (en) * | 2008-11-14 | 2010-05-20 | 三井化学株式会社 | Nitrile hydratase variant |
| CN102492016A (en) * | 2011-11-25 | 2012-06-13 | 江南大学 | Method for separation purification of nitrile hydratase regulatory protein |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010055666A1 (en) * | 2008-11-14 | 2010-05-20 | 三井化学株式会社 | Nitrile hydratase variant |
| CN102492016A (en) * | 2011-11-25 | 2012-06-13 | 江南大学 | Method for separation purification of nitrile hydratase regulatory protein |
Non-Patent Citations (3)
| Title |
|---|
| KAMILA RZEZNICKA等: "Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli,its purification and biochemical characterisation", 《APPL MICROBIOL BIOTECHNOL》, vol. 85, 31 December 2010 (2010-12-31), pages 1417 - 1425 * |
| LINKE B: "AM902716.1", 《EMBL BANK》, 28 August 2008 (2008-08-28), pages 1 * |
| 史悦 等: "腈水合酶基因克隆与调控表达的研究进展", 《中国生物工程杂志》, vol. 24, no. 7, 31 December 2004 (2004-12-31), pages 34 - 39 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020147031A1 (en) * | 2019-01-16 | 2020-07-23 | 江南大学 | Nitrile hydratase mutant, genetically engineered bacterium containing same, and use thereof |
| US11332731B2 (en) | 2019-01-16 | 2022-05-17 | Jiangnan University | Nitrile hydratase mutant, genetically engineered bacterium containing mutant and applications thereof |
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Application publication date: 20120613 |