CN105219821A - The method of exoprotein is synthesized in a kind of microcapsule of alginate composition - Google Patents
The method of exoprotein is synthesized in a kind of microcapsule of alginate composition Download PDFInfo
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- CN105219821A CN105219821A CN201510720025.3A CN201510720025A CN105219821A CN 105219821 A CN105219821 A CN 105219821A CN 201510720025 A CN201510720025 A CN 201510720025A CN 105219821 A CN105219821 A CN 105219821A
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Abstract
The invention discloses the method for synthesizing exoprotein in the microcapsule of a kind of alginate composition, take following steps: inject outer water phase, oil phase and internal layer aqueous phase in microfluidic devices respectively; Regulate the osmotic pressure of internal layer aqueous phase, form water-in-oil-in-water drop at the Nodes of microchannel; It contacts with crosslinker solution by slide glass; Exoprotein is being synthesized with the enzyme in intestinal bacteria test kit and substrate; By the semi-permeable microcapsule of collected by centrifugation.By above preparation method, the semi-transparent polyion complex compound film that the alginate applied by polymine form can make the substrate of synthetic protein be diffused into smoothly in microcapsule, and substrate and by product continue exchange by film can make protein continue synthesis; Although the little protein making to synthesize of the volume due to microcapsule is little, small molecule by-product can pass through semipermeable partition and secretes away, and protein remains is retained in microcapsule.
Description
Technical field
The invention belongs to field of biological pharmacy, particularly relate to a kind of method of synthesizing exoprotein in semi-transparent microcapsule be made up of polymine coating alginate.
Background technology
The function understanding protein from molecular level studies the key of biological gene, nowadays along with the development of biotechnology, more and more higher to the requirement of protein expression purification technique, but the cell recombinant protein plastome of at present tradition application cannot make research reach perfect condition, so cell-free protein system is arisen at the historic moment and is rapidly developed because of himself limitation and deficiency.
Cell-free protein synthesis system be a kind of with mRNA or DNA for template, carry out the vitro system of synthetic protein by supplementing substrate and energy substance in the enzyme system of cell extract.This protein synthesis system can synthesize target protein not having active somatic cell to deposit to carry out in case continuous print to transcribe and translate.Compared with traditional In vivo recombination expression system, the advantage that external cell free translation system has it special: 1, in most energy and born of the same parents resource for the synthesis of target protein; 2, the condition phase commute control of protein synthesis, can conveniently add required substrate and catalyzer, promotes the expression of albumen and folds; 3, for expressing poisonous protein, avoid toxic protein to the lethal effect of host cell; 4, in the upper parallel projects multiple proteins simultaneously of porous plate (as 96 orifice plates), the demand of high-flux medicaments sifting and proteomics research can be met; 5, directly using PCR primer as templated synthesis protein, can carry out the rapid screening of mutain, realize the lactam enzyme by directional anagenesis in vitro of protein molecular; 6, reaction system is flexible, can with other biotechnology phase coupling, target protein purifying is convenient, is conducive to conducting a research of subsequent step.
Cell-free protein synthesis system is applied to (1) structural proteomics; (2) high flux screening and functional proteomics; (3) protein evolution; (4) special marking is carried out to protein; (5) microminiaturization of protein synthesis, cell-free protein synthesis system even can reach the yardstick received and rise, and this is that body internal protein synthesizes the level that cannot imagine.But cell free system can not carry out common translation and post-translational glycosylation is modified, the complexity can not carrying out other eukaryotic protein is modified.Although cell free, protein synthesizing system test kit is easy to use, the energy in expensive and system and substrate all need constantly to regenerate, and also cannot be generalized to suitability for industrialized production at present.Therefore require further study to reduce costs.
Summary of the invention
In order to solve the problem, the invention provides the method for synthesizing exoprotein in the microcapsule of a kind of alginate composition, to improve the deficiencies in the prior art.
For this reason, adopt following technical scheme, synthesize the method for exoprotein in a kind of microcapsule of alginate composition, it is characterized in that taking following steps:
A) inject outer water phase, oil phase and internal layer aqueous phase in microfluidic devices respectively, flow velocity is respectively 0.01-0.05,0.1-0.3 and 4-5mol/L; Described outer water phase is the ultrapure water containing 1.0wt% empgen BB, and described internal layer aqueous phase is the mixed solution of the fluorescein dextran containing the ultrapure water of 1.0wt% sodium alginate, the sodium-chlor of 0.6wt% and 0.1wt%;
B) regulate the osmotic pressure of internal layer aqueous phase to 200-500, form water-in-oil-in-water drop at the Nodes of microchannel;
C) on slide glass by step B) the water-in-oil-in-water emulsion that obtains with comprise the crosslinker solution of 0.05-0.5mol/L calcium chloride with 0.05-0.5wt% polymine and contact, formed the alginate calcium globule of band polyion composite membrane by crosslinking reaction;
D) with the enzyme in intestinal bacteria test kit and substrate synthesis exoprotein, with the DNA encoding of the green fluorescent protein in test kit for template, DNA concentration is 0.07-2ng/ μ L;
E) template DNA, transcriptase and translation enzyme and substrate are added steps A) in internal layer aqueous phase, carry out above-mentioned steps A, B, C successively, obtain semi-permeable microcapsule;
F) by collected by centrifugation step e) the semi-permeable microcapsule that obtain, aqueous phase containing substrate is added in microcapsule suspensions, microcapsule suspensions is placed in culture dish, cultivate at 20-50 DEG C and make protein synthesis in 2-10 hour, then cultivate at 5-10 DEG C and protein was fully formed in 12-24 hour.
By above preparation method, the invention has the beneficial effects as follows: (1) microcapsule are prepared by the induction method for packing that breaks by water-in-oil-in-water droplet, can reduce the seepage of packed material, and obtained microcapsule have unified size distribution; (2) oil-in-water droplet is divided into the enzyme needed for synthesis exoprotein by microfluidic device etc. with the template DNA aqueous solution of green fluorescent protein coding, droplet is converted into semi-permeable microcapsule, is confirmed the synthesis of exoprotein by the fluorescence detected in microcapsule in the egfp that synthesizes; (3) the semi-transparent polyion complex compound film that the alginate applied by polymine form can make the substrate of synthetic protein be diffused into smoothly in microcapsule, and substrate and by product continue exchange by film can make protein continue synthesis; (4) although make the protein that synthesizes little because the volume of microcapsule is little, small molecule by-product can pass through semipermeable partition and secretes away, and protein remains is retained in microcapsule.
Embodiment
Embodiment 1 takes following steps:
A) inject outer water phase, oil phase and internal layer aqueous phase in microfluidic devices respectively, flow velocity is respectively 0.01, and 0.1 and 4mol/L; Described outer water phase is the ultrapure water containing 1.0wt% empgen BB, and described internal layer aqueous phase is the mixed solution of the fluorescein dextran containing the ultrapure water of 1.0wt% sodium alginate, the sodium-chlor of 0.6wt% and 0.1wt%;
B) regulate the osmotic pressure to 200 of internal layer aqueous phase, form water-in-oil-in-water drop at the Nodes of microchannel;
C) on slide glass by step B) the water-in-oil-in-water emulsion that obtains with comprise the crosslinker solution of 0.05mol/L calcium chloride with 0.05wt% polymine and contact, formed the alginate calcium globule of band polyion composite membrane by crosslinking reaction;
D) with the enzyme in intestinal bacteria test kit and substrate synthesis exoprotein, with the DNA encoding of the green fluorescent protein in test kit for template, DNA concentration is 0.07ng/ μ L;
E) template DNA, transcriptase and translation enzyme and substrate are added steps A) in internal layer aqueous phase, carry out above-mentioned steps A, B, C successively, obtain semi-permeable microcapsule;
F) by collected by centrifugation step e) the semi-permeable microcapsule that obtain, aqueous phase containing substrate is added in microcapsule suspensions, microcapsule suspensions is placed in culture dish, cultivates at 20 DEG C and make protein synthesis in 10 hours, then cultivate at 5 DEG C and protein was fully formed in 24 hours.
Embodiment 2 takes following steps:
A) inject outer water phase, oil phase and internal layer aqueous phase in microfluidic devices respectively, flow velocity is respectively 0.03, and 0.2 and 4.5mol/L; Described outer water phase is the ultrapure water containing 1.0wt% empgen BB, and described internal layer aqueous phase is the mixed solution of the fluorescein dextran containing the ultrapure water of 1.0wt% sodium alginate, the sodium-chlor of 0.6wt% and 0.1wt%;
B) regulate the osmotic pressure to 350 of internal layer aqueous phase, form water-in-oil-in-water drop at the Nodes of microchannel;
C) on slide glass by step B) the water-in-oil-in-water emulsion that obtains with comprise the crosslinker solution of 0.3mol/L calcium chloride with 0.3wt% polymine and contact, formed the alginate calcium globule of band polyion composite membrane by crosslinking reaction;
D) with the enzyme in intestinal bacteria test kit and substrate synthesis exoprotein, with the DNA encoding of the green fluorescent protein in test kit for template, DNA concentration is 1ng/ μ L;
E) template DNA, transcriptase and translation enzyme and substrate are added steps A) in internal layer aqueous phase, carry out above-mentioned steps A, B, C successively, obtain semi-permeable microcapsule;
F) by collected by centrifugation step e) the semi-permeable microcapsule that obtain, aqueous phase containing substrate is added in microcapsule suspensions, microcapsule suspensions is placed in culture dish, cultivates at 35 DEG C and make protein synthesis in 6 hours, then cultivate at 5 DEG C and protein was fully formed in 18 hours.
Embodiment 3 takes following steps:
A) inject outer water phase, oil phase and internal layer aqueous phase in microfluidic devices respectively, flow velocity is respectively 0.05, and 0.3 and 5mol/L; Described outer water phase is the ultrapure water containing 1.0wt% empgen BB, and described internal layer aqueous phase is the mixed solution of the fluorescein dextran containing the ultrapure water of 1.0wt% sodium alginate, the sodium-chlor of 0.6wt% and 0.1wt%;
B) regulate the osmotic pressure to 500 of internal layer aqueous phase, form water-in-oil-in-water drop at the Nodes of microchannel;
C) on slide glass by step B) the water-in-oil-in-water emulsion that obtains with comprise the crosslinker solution of 0.5mol/L calcium chloride with 0.5wt% polymine and contact, formed the alginate calcium globule of band polyion composite membrane by crosslinking reaction;
D) with the enzyme in intestinal bacteria test kit and substrate synthesis exoprotein, with the DNA encoding of the green fluorescent protein in test kit for template, DNA concentration is 2ng/ μ L;
E) template DNA, transcriptase and translation enzyme and substrate are added steps A) in internal layer aqueous phase, carry out above-mentioned steps A, B, C successively, obtain semi-permeable microcapsule;
F) by collected by centrifugation step e) the semi-permeable microcapsule that obtain, aqueous phase containing substrate is added in microcapsule suspensions, microcapsule suspensions is placed in culture dish, cultivates at 50 DEG C and make protein synthesis in 2 hours, then cultivate at 10 DEG C and protein was fully formed in 12 hours.
Protection scope of the present invention is not limited to above several embodiment, and therefore, every technical scheme formed by simple numerical value replacement etc., is all formed specific embodiments of the invention, and form protection scope of the present invention.
Claims (1)
1. synthesize a method for exoprotein in the microcapsule that alginate form, it is characterized in that taking following steps:
A) inject outer water phase, oil phase and internal layer aqueous phase in microfluidic devices respectively, flow velocity is respectively 0.01-0.05,0.1-0.3 and 4-5mol/L; Described outer water phase is the ultrapure water containing 1.0wt% empgen BB, and described internal layer aqueous phase is the mixed solution of the fluorescein dextran containing the ultrapure water of 1.0wt% sodium alginate, the sodium-chlor of 0.6wt% and 0.1wt%;
B) regulate the osmotic pressure of internal layer aqueous phase to 200-500, form water-in-oil-in-water drop at the Nodes of microchannel;
C) on slide glass by step B) the water-in-oil-in-water emulsion that obtains with comprise the crosslinker solution of 0.05-0.5mol/L calcium chloride with 0.05-0.5wt% polymine and contact, formed the alginate calcium globule of band polyion composite membrane by crosslinking reaction;
D) with the enzyme in intestinal bacteria test kit and substrate synthesis exoprotein, with the DNA encoding of the green fluorescent protein in test kit for template, DNA concentration is 0.07-2ng/ μ L;
E) template DNA, transcriptase and translation enzyme and substrate are added steps A) in internal layer aqueous phase, carry out above-mentioned steps A, B, C successively, obtain semi-permeable microcapsule;
F) by collected by centrifugation step e) the semi-permeable microcapsule that obtain, aqueous phase containing substrate is added in microcapsule suspensions, microcapsule suspensions is placed in culture dish, cultivate at 20-50 DEG C and make protein synthesis in 2-10 hour, then cultivate at 5-10 DEG C and protein was fully formed in 12-24 hour.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020255108A1 (en) * | 2019-06-20 | 2020-12-24 | Vilnius University | Systems and methods for encapsulation and multi-step processing of biological samples |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1131014A (en) * | 1994-12-06 | 1996-09-18 | 赫里尼·柯蒂斯公司 | Water-in-oil-in-water compositions |
| CN101092446A (en) * | 2000-08-29 | 2007-12-26 | 株式会社无细胞科学 | Methods of synthesizing cell-free protein |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1131014A (en) * | 1994-12-06 | 1996-09-18 | 赫里尼·柯蒂斯公司 | Water-in-oil-in-water compositions |
| CN101092446A (en) * | 2000-08-29 | 2007-12-26 | 株式会社无细胞科学 | Methods of synthesizing cell-free protein |
Non-Patent Citations (2)
| Title |
|---|
| D. SAEKI等: "MICROCOMPARTMENTALIZED CELL-FREE PROTEIN SYNTHESIS FROM SINGLE MOLECULE TEMPLATE DNA USING SEMI-PERMEABLE ALGINATE MICROCAPSULES", 《15TH INTERNATIONAL CONFERENCE ON MINIATURIZED SYSTEMS FOR CHEMISTRY AND LIFE SCIENCES》 * |
| 侯利华: "无细胞蛋白合成系统的研究进展", 《国外医学分子生物学分册》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020255108A1 (en) * | 2019-06-20 | 2020-12-24 | Vilnius University | Systems and methods for encapsulation and multi-step processing of biological samples |
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