CN102586312B - Method for expressing intracellular protein matrix and application thereof - Google Patents
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention discloses a method for expressing intracellular protein matrix and application thereof, belonging to the technical field of biological engineering. Co-expression of T.fusca cutinase and intracellular target protein in Escherichia Coli is realized; therefore, extracellular expression of the target protein is achieved. The enzymatic activity of fermentation culture supernatant can be more than 80 percent of the total enzymatic activity, and the subsequent extraction process can be simplified. The method has higher academic significance and application value.
Description
Technical field
The present invention relates to a kind of method that intracellular protein matrix is expressed and its application, belong to technical field of bioengineering.
Background technology
According to locator means, gene engineering expression foreign protein is generally divided into two kinds of forms: intracellular expression after target protein is synthetic in tenuigenin rrna, is still positioned in born of the same parents under a series of chaperonins help; Extracellular expression, secretes to born of the same parents by the functional sequence of albumen itself and the processing transport system of Host Strains after target protein is synthetic.Exocytosis type is expressed can simplify downstream purification process greatly, cost-saving, therefore in large-scale industrial production, has absolute predominance.
Intestinal bacteria (Escherichia coli) genetic background is clear, and culture cycle is short, simple to operate, is current most widely used general, the most successful external source expression of recombinant proteins system.5 kinds of Secretory Pathways of the natural existence of intestinal bacteria, various Secretory Pathways all have its specific translocator and mechanism of secretion, and recombinant protein is all had to unique requirement, in general, only under native state, when recombinant expressed, just likely realize matrix secretion type for extracellular enzyme and express, be transported to extracellular report and have no intracellular protein by the excretory system of Host Strains.
Colibacillary cytolemma has two-layer, inner membrance is made up of phosphatide and membranin, wherein phosphatide comprises the phosphatidylethanolamine (phosphatidyl-ethanolamine) of 70-80%, the phosphatidyl glycerol (phosphatidylglycerol) of 15-20% and lower than 5% Val (cardiolipin), saturated fatty acid and phosphatidylethanolamine are rich in adventitia inner side, and outside is mainly lipopolysaccharides.Increase artificially the permeability of cytolemma, can accelerate the rate of discharge of intracellular product, be beneficial to the accumulation of target protein in substratum.The method that cell membrane permeability regulates and controls is a lot, from genetic angle, can select permeability of cell membrane mutant strain; From rear period regulation angle, according to the difference of material transmembrane transport mechanism, can take corresponding method.At present widely used is chemical method, in substratum, adds such as penicillin, Mn
2+, tensio-active agent, Na
+, N.F,USP MANNITOL etc., can destroy to a certain extent the integrity of cytolemma, improve fermentation yield.
At is a kind of multifunctional enzyme, the various solubility ester of hydrolyzable class, the triglyceride level of emulsification, insoluble fatty acid ester, insoluble polymer cutin etc., the at of the currently reported F.solani of deriving from has the hydrolytic activity of phosphatidylethanolamine (main component of membrane phospholipid in intestinal bacteria, the integral part of adventitia).The permeability that therefore can improve cytolemma to the hydrolytic action of Bacillus coli cells membrane phospholipid composition by is expressed born of the same parents' internal object albumen substrate.
The present invention chooses two kinds of important industrial enzymes beta-galactosidase enzymess and isoamylase as reporter protein.Beta-galactosidase enzymes is a kind of multifunctional enzyme, can act on β (1 → 4) glycosidic link of lactose, forms galactosyl-enzyme complex, and under the hydrolytic activity of this enzyme, mixture is further hydrolyzed, and generates semi-lactosi; And under the glycosides vigor that the turns effect of this enzyme, mixture further with the combination such as monose, disaccharides or trisaccharide, generate oligomeric galactose.Beta-galactosidase enzymes is the main with enzyme of industrial Production by Enzymes new type functional oligose oligomeric galactose, and the market requirement is large; Isoamylase can cut α-1 in amylopectin tapping point in specific manner, and 6 glycosidic links are cut whole side shoot, generate amylose starch different in size.Multiple starch deep processing fields and the field of medicaments such as starch syrup, beer and Alcohol Production are widely used at present.
Summary of the invention
The invention provides a kind of method that intracellular protein matrix is expressed, AAZ54921) and born of the same parents' internal object protein gene coexpression in Host Strains intestinal bacteria be by T.fusca at gene (NCBI numbering:, through fermentation culture, collect fermented supernatant fluid.
For solving the problems of the technologies described above, concrete grammar is:
1) pETDuet-1 plasmid and Tfu_0883/pET20b (+) are carried out to double digestion, ligase enzyme connection is spent the night, and connects product Transformed E .coli JM109 competent cell, obtains recombinant plasmid Tfu_0883/pETDuet-1;
2) Tfu_0883/pETDuet-1 and the recombinant plasmid pET20b (+) that contains born of the same parents' internal object protein gene are carried out to double digestion, ligase enzyme connection is spent the night, connect product Transformed E .coli JM109 competent cell, obtain the recombinant plasmid Tfu_0883/pETDuet-1 that contains target protein gene;
3) the recombinant plasmid Tfu_0883/pETDuet-1 that contains target protein gene is imported to host e. coli;
4) step 3) build recombination bacillus coli target fermentation product albumen, collect fermented supernatant fluid.
Described Host Strains can be: E.coli BL21 (DE3), E.coliW3110, E.coliJM109, E.coliJM109 (DE3), E.coliDH5 α etc., wherein preferred E.coli BL21 (DE3).
The described carrier that carries at and target protein is the double-promoter carriers such as pETDuet-1, pCOLADuet-1; PUC series, pET series, pT7-7, pGEX etc. are any.
Described born of the same parents' internal object albumen refers to that target protein is at intracellular expression, after it is synthetic in tenuigenin rrna, under a series of chaperonins help, is still positioned in born of the same parents; For example: beta-galactosidase enzymes (NCBI numbering: AE006641) and isoamylase (NCBI numbers: NC_007333).Beta-galactosidase enzymes is a kind of multifunctional enzyme, can act on β (1 → 4) glycosidic link of lactose, forms galactosyl-enzyme complex, and under the hydrolytic activity of this enzyme, mixture is further hydrolyzed, and generates semi-lactosi; And under the glycosides vigor that the turns effect of this enzyme, mixture further with the combination such as monose, disaccharides or trisaccharide, generate oligomeric galactose.Beta-galactosidase enzymes is the main with enzyme of industrial Production by Enzymes new type functional oligose oligomeric galactose, and the market requirement is large; Isoamylase can cut α-1 in amylopectin tapping point in specific manner, and 6 glycosidic links are cut whole side shoot, generate amylose starch different in size.Multiple starch deep processing fields and the field of medicaments such as starch syrup, beer and Alcohol Production are widely used at present.
Construction process containing at recombinant plasmid is: pETDuet-1 plasmid (purchased from Novagen company) and Tfu 0883/pET20b (+) are carried out to double digestion, spend the night with 16 DEG C of connections of T4 ligase enzyme, connect product Transformed E .coli JM109 competent cell, select transformant and extract plasmid.
Described Tfu_0883/pET20b (+) (Chen S, Tong X, Woodard RW, Du GC, Wu J, Chen J, Identification and Characterization of Bacterial Cutinase, The Journal of Biological Chemistry, 2008,283 (28) 25854-25862) for containing T.fusca at gene (NCBI numbering: recombinant plasmid AAZ54921).
The expression of born of the same parents' internal object albumen: carry out double digestion containing at recombinant plasmid and the recombinant plasmid containing born of the same parents' internal object protein gene by above-mentioned, ligase enzyme connects, and Transformed E .coli JM109 competent cell, selects transformant and extract recombinant plasmid, imported Host Strains, carried out liquid culture.
The present invention is by coexpression T.fusca at, and Bacillus coli cells permeability improves, and target protein enzyme work in substratum can reach the more than 80% of total enzyme work, makes intracellular protein realize matrix and expresses.
Embodiment
Embodiment 1:
The structure of Tfu_0883/pETDuet-1 recombinant plasmid: the Tfu_0883/pET20b (+) of pETDuet-1 plasmid and laboratory structure in early stage is carried out to NcoI and HandIII double digestion, enzyme is cut after product rubber tapping recovery, spend the night with 16 DEG C of connections of T4 ligase enzyme, connect product Transformed E .coli JM109 competent cell, converted product coating is containing the LB solid plate of 100mg/L penbritin, through 37 DEG C of overnight incubation, selecting transformant cultivates in the LB liquid nutrient medium containing 100mg/L penbritin, then extract plasmid, obtain recombinant plasmid Tfu_0883/pETDuet-1.
The structure of Tfu_0883/lacS/pETDuet-1 recombinant plasmid: lacS/pET20b (+) (Wu Jing of Tfu_0883/pETDuet-1 plasmid and laboratory structure in early stage, Chen Sheng, Wu Yufei, Chen Jian, Xia Zehua: mutant of a kind of beta-galactosidase enzymes and its preparation method and application .201110247338.3) carry out NdeI and XhoI double digestion, enzyme is cut after product rubber tapping recovery, spend the night with 16 DEG C of connections of T4 ligase enzyme again, connect product Transformed E .coli JM109 competent cell, converted product coating is containing the LB solid plate of 100mg/L penbritin, through 37 DEG C of overnight incubation, selecting transformant cultivates in the LB liquid nutrient medium containing 100mg/L penbritin, then extract plasmid, obtain recombinant plasmid Tfu_0883/lacS/pETDuet-1.
The expression of beta-galactosidase enzymes and preparation: by Tfu_0883/lacS/pETDuet-1 Transformed E .coli BL21 (DE3) Host Strains, containing on the LB solid plate of 100mg/L penbritin through 37 DEG C of cultivation 8-10h, select transformant 37 DEG C of cultivation 8-10h in LB liquid nutrient medium, after with 5% inoculum size access TB (glycerine 5g/L, peptone 12g/L, yeast extract paste 24g/L, K
2hPO
412.54g/L, KH
2pO
42.31g/L) after 37 DEG C of cultivation 2h of fermentation broth (containing 100 μ g/mL penbritins), induce about 16h with 1mM IPTG (isopropylthio-β-D galactoside).
Fermented liquid is in 4 DEG C, the centrifugal 10min of 8000rpm, the beta-galactosidase enzymes enzyme of measuring respectively in fermented liquid supernatant and cell is lived as 102U/mL and 17U/mL, extracellular enzyme work account for that total enzyme lives 85.4%.
Embodiment 2:
The structure of Tfu_0883/Tfu_1891/pETDuet-1 recombinant plasmid: by Tfu_1891/pET20b (+) (Wu Jing of Tfu_0883/pETDuet-1 plasmid and laboratory structure in early stage, Chen Sheng, Duan Xuguo, Chen Jian: a kind of acidic heat-resisting isoamylase genetic engineering bacterium and application .201110459137.X thereof) carry out NdeI and XhoI double digestion, enzyme is cut after product rubber tapping recovery, spend the night with 16 DEG C of connections of T4 ligase enzyme again, connect product Transformed E .coli JM109 competent cell, converted product coating is containing the LB solid plate of 100mg/L penbritin, through 37 DEG C of overnight incubation, selecting transformant cultivates in the LB liquid nutrient medium containing 100mg/L penbritin, then extract plasmid, obtain recombinant plasmid Tfu_0883/Tfu_1891/pETDuet-1.
The expression of isoamylase and preparation: by Tfu_0883/Tfu_1891/pETDuet-1 Transformed E .coli BL21 (DE3) Host Strains, containing on the LB solid plate of 100mg/L penbritin through 37 DEG C of cultivation 8-10h, select transformant 37 DEG C of cultivation 8-10h in LB liquid nutrient medium, after with 5% inoculum size access TB (glycerine 5g/L, peptone 12g/L, yeast extract paste 24g/L, K
2hPO
412.54g/L, KH
2pO
42.31g/L) after 37 DEG C of cultivation 2h of fermentation broth (containing 100 μ g/mL penbritins), induce about 16h with 1mM IPTG (isopropylthio-β-D galactoside).
Fermented liquid is in 4 DEG C, the centrifugal 10min of 8000rpm, the isoamylase enzyme of measuring respectively in fermented liquid supernatant and cell is lived as 733U/mL and 142U/mL, extracellular enzyme work account for that total enzyme lives 83.8%.
Claims (5)
1. the method that intracellular protein matrix is expressed, it is characterized in that, NCBI is numbered to the at gene of AAZ54921 and born of the same parents' internal object protein gene at Host Strains expression in escherichia coli, fermentation culture is also collected fermented supernatant fluid, and described born of the same parents' internal object albumen is the protein at cell inner expression; Concrete grammar is:
1) pETDuet-1 plasmid and Tfu_0883/pET20b (+) are carried out to double digestion, ligase enzyme connection is spent the night, and connects product Transformed E .coli JM109 competent cell, obtains recombinant plasmid Tfu_0883/pETDuet-1;
2) Tfu_0883/pETDuet-1 and the recombinant plasmid pET20b (+) that contains born of the same parents' internal object protein gene are carried out to double digestion, ligase enzyme connection is spent the night, connect product Transformed E .coli JM109 competent cell, obtain the recombinant plasmid Tfu_0883/pETDuet-1 that contains target protein gene;
3) the recombinant plasmid Tfu_0883/pETDuet-1 that contains target protein gene is imported to host e. coli;
4) the recombination bacillus coli target fermentation product albumen that step 3) builds, collects fermented supernatant fluid; Described Tfu_0883 is the at gene that NCBI is numbered AAZ54921.
2. method claimed in claim 1, is characterized in that, described host e. coli is any in E.coli BL21 (DE3), E.coli W3110, E.coli JM109, E.coli JM109 (DE3), E.coli DH5 α.
3. method claimed in claim 2, is characterized in that, described host e. coli is E.coli BL21 (DE3).
4. method claimed in claim 1, is characterized in that, described in contain born of the same parents' internal object protein gene recombinant plasmid pET20b (+) be lacS/pET20b (+) or Tfu_1891/pET20b (+); Described Tfu_1891 is the heat-resisting isoamylase gene of acidity that NCBI is numbered NC_007333.1.
5. the application of method in intracellular protein matrix is expressed described in a claim 1.
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| CN108753671A (en) * | 2018-06-05 | 2018-11-06 | 江南大学 | A kind of the recombination bacillus coli engineering bacteria and its zymotechnique of high yield cutinase |
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| CN102827863A (en) * | 2012-09-03 | 2012-12-19 | 江南大学 | Method for accelerating extracellular protein matrix secretion and application thereof |
| CN104878033B (en) * | 2015-04-29 | 2018-08-28 | 江南大学 | A kind of method and its application promoting target protein extracellular expression |
| CN106754601A (en) * | 2016-12-21 | 2017-05-31 | 江南大学 | A kind of application phospholipase C by intracellular protein extracellular expression method |
| CN109679973A (en) * | 2018-12-25 | 2019-04-26 | 深圳市刚竹医疗科技有限公司 | Archaeal dna polymerase and preparation method thereof, expressing gene, expression vector, host cell and kit |
| CN111117940B (en) * | 2019-12-04 | 2022-06-28 | 天津大学 | Escherichia coli engineering bacterium and method for high yield of pentamethylene diamine |
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| CN101792729A (en) * | 2009-12-18 | 2010-08-04 | 江南大学 | Genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and method for constructing same |
| CN102080063A (en) * | 2010-12-08 | 2011-06-01 | 江南大学 | Cutinase producing gene engineering bacteria and use thereof |
| CN102337254A (en) * | 2011-08-26 | 2012-02-01 | 江南大学 | Mutant of beta-galactosidase and preparation method and application thereof |
| CN102559568A (en) * | 2011-12-31 | 2012-07-11 | 江南大学 | Acidic heat-resisting isoamylase genetic engineering bacterium and application thereof |
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2012
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Patent Citations (6)
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| CN101591648A (en) * | 2009-06-05 | 2009-12-02 | 江南大学 | The preparation of a kind of heat resistance cutinase-CBD and the application in cotton fiber refining thereof |
| CN101792729A (en) * | 2009-12-18 | 2010-08-04 | 江南大学 | Genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and method for constructing same |
| CN101705211A (en) * | 2009-12-21 | 2010-05-12 | 江南大学 | Process for fermentation production of recombined cutinase |
| CN102080063A (en) * | 2010-12-08 | 2011-06-01 | 江南大学 | Cutinase producing gene engineering bacteria and use thereof |
| CN102337254A (en) * | 2011-08-26 | 2012-02-01 | 江南大学 | Mutant of beta-galactosidase and preparation method and application thereof |
| CN102559568A (en) * | 2011-12-31 | 2012-07-11 | 江南大学 | Acidic heat-resisting isoamylase genetic engineering bacterium and application thereof |
Cited By (1)
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| CN108753671A (en) * | 2018-06-05 | 2018-11-06 | 江南大学 | A kind of the recombination bacillus coli engineering bacteria and its zymotechnique of high yield cutinase |
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