CN107299058A - A kind of cell extract synthesized for cell-free protein and preparation method thereof - Google Patents
A kind of cell extract synthesized for cell-free protein and preparation method thereof Download PDFInfo
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- CN107299058A CN107299058A CN201710599126.9A CN201710599126A CN107299058A CN 107299058 A CN107299058 A CN 107299058A CN 201710599126 A CN201710599126 A CN 201710599126A CN 107299058 A CN107299058 A CN 107299058A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/063—Lysis of microorganisms of yeast
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
Abstract
The invention discloses a kind of cell extract synthesized for cell-free protein and preparation method thereof, method is：(1) yeast extract powder peptone glucose culture solution culture yeasts cell is used, yeast cells is collected in separation；(2) yeast cells collected is washed 25 times using lavation buffer solution, and is suspended again with disruption buffer, is crushed；Centrifuge, supernatant is to be used for the cell extract that cell-free protein is synthesized.It is used for the purposes of external Fast back-projection algorithm protein for the cell extract that cell-free protein is synthesized.Preparation method of the present invention is easy and effective, and step is few, saves cumbersome dialysis procedure, and the cell extract of acquisition is synthesized available for external cell-free protein.
Description
Technical field
The present invention relates to a kind of cell extract and Preparation method and use synthesized for cell-free protein, belong to raw
Thing synthesizes field.
Background technology
Cell-free protein synthetic system is a kind of using external source messenger RNA or DNA as template, thin
Substrate is supplemented in born of the same parents' extract and energy carrys out the vitro system of synthetic protein, one that protein field is had become at present grinds greatly
Study carefully focus.The preparation of wherein cell extract also develops into a variety of cells from initial Escherichia coli.Yeast is used as one
Kind of eukaryotic, genetic background is clear, and growth is quick and cheap, for cell-free protein synthesis, with production is quick, yield
It is high, can produce and need the advantage of posttranslational modification albumen.There is presently no the yeast cells synthesized for cell-free protein extraction
The report of the easy and effective preparation method of thing.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of cell synthesized for cell-free protein
Extract.
Second object of the present invention is to provide a kind of preparation side of the cell extract synthesized for cell-free protein
Method.
Third object of the present invention is to provide a kind of purposes of the cell extract synthesized for cell-free protein.
The solution of the present invention is summarized as follows：
A kind of preparation method of the cell extract synthesized for cell-free protein, comprises the following steps：
(1) yeast extract powder peptone glucose culture solution culture yeasts cell is used, yeast cells is collected in separation；
(2) yeast cells collected is washed 2-5 times using lavation buffer solution, and is suspended again with disruption buffer, is carried out
It is broken；Centrifuge, supernatant is to be used for the cell extract that cell-free protein is synthesized.
The composition of yeast extract powder peptone glucose culture solution：10-30g/L yeast extracts, 5-15g/L peptones, 25-
35g/L glucose, solvent is deionized water.
The condition of culture yeasts cell is：25 DEG C -32 DEG C of temperature, rotating speed are 150rpm-250rpm, and the time is 3-18h.
The composition of lavation buffer solution：PH=7.2-7.6 20-50mmol/L4- hydroxyethyl piperazineethanesulfonic acids-potassium hydroxide,
80-120mmol/L potassium glutamates, 1-4mmol/L psicosomas, 1-4mmol/L dithiothreitol (DTT)s, 50g/L-100g/L sweet dews
Alcohol；Solvent is deionized water.
The composition of disruption buffer：PH=7.2-7.6 20-50mmol/L 4- hydroxyethyl piperazineethanesulfonic acids-hydroxide
Potassium, 80-120mmol/L potassium glutamates, 1-4mmol/L psicosomas, 1-4mmol/L dithiothreitol (DTT)s, 50g/L-100g/L is sweet
Reveal alcohol, 0.2-0.8mmol/L phenylmethylsulfonyl fluorides；Solvent is deionized water.
Step (2) the broken method is Mechanical Method, high-pressure homogenization, sonioation method or glass bead method.
The rotating speed of step (2) described centrifugation is 20000 × g-30000 × g.
Prepared by the above method is used for the cell extract that cell-free protein is synthesized.
It is used for the purposes of external Fast back-projection algorithm protein for the cell extract that cell-free protein is synthesized.
Advantages of the present invention：
It is an advantage of the invention that preparation method is easy and effective, step is few, saves cumbersome dialysis procedure, and the cell of acquisition is carried
Thing is taken to be synthesized available for external cell-free protein.
Brief description of the drawings
Fig. 1 is embodiment 10, embodiment 11, the cell extract synthesized for cell-free protein of the preparation of embodiment 12
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis figure.
Swimming lane 1 is the cell extract synthesized for cell-free protein prepared by embodiment 10, and swimming lane 2 is embodiment 11
What is prepared is used for the cell extract that cell-free protein is synthesized, and swimming lane 3 is the preparation of embodiment 12 for cell-free protein
The cell extract of synthesis.
Fig. 2 is the fractional yield using the external Fast back-projection algorithm protein of cell extract for being used for cell-free protein synthesis
Figure.
Embodiment
Below by specific embodiment, the present invention is further illustrated.The following examples are in order that this area
Technical staff better understood when the present invention, but the present invention not imposed any restrictions.
The yeast cells source that various embodiments of the present invention are used：The saccharomyces cerevisiae BY4741 of ATCC 4040002
https://www.atcc.org/Products/All/201388.aspx
The composition of yeast extract powder peptone glucose culture solution is shown in Table 1 embodiment 1-3
Table 1
| Embodiment 1 | Embodiment 2 | Embodiment 3 | |
| Yeast extract | 10g/L | 20g/L | 30g/L |
| Peptone | 5g/L | 10g/L | 15g/L |
| Glucose | 25g/L | 30g/L | 35g/L |
| Solvent is deionized water | Complement to 1L | Complement to 1L | Complement to 1L |
Lavation buffer solution composition is shown in Table 2 embodiment 4-6
Table 2
Disruption buffer composition is shown in Table 3 embodiment 7-9
Table 3
Embodiment 10
A kind of preparation method of the cell extract synthesized for cell-free protein, comprises the following steps：
(1) using the yeast extract powder peptone glucose culture solution of embodiment 1, temperature be 25 DEG C, rotating speed be 150rpm, training
The time for supporting yeast cells is 3h；Separation, collects yeast cells；
(2) yeast cells collected washs yeast cells with the lavation buffer solution of embodiment 42 times, and breaking with embodiment 7
Broken buffer solution is resuspended；Crushed with Mechanical Method, be that 20000 × g is centrifuged in rotating speed, supernatant is to be used for without thin
The cell extract of born of the same parents' protein synthesis.
Embodiment 11
A kind of preparation method of the cell extract synthesized for cell-free protein, comprises the following steps：
(1) using the yeast extract powder peptone glucose culture solution of embodiment 2, temperature be 28 DEG C, rotating speed be 200rpm, training
It is 10h to support the yeast cells time；Separation, collects yeast cells；
(2) yeast cells is washed with the lavation buffer solution of embodiment 53 times after collecting cell, and is delayed with the broken of embodiment 8
Fliud flushing is resuspended, and is crushed with high-pressure homogenization, is that 25000 × g is centrifuged in rotating speed, supernatant is for acellular
The cell extract of protein synthesis.
Embodiment 12
A kind of preparation method of the cell extract synthesized for cell-free protein, comprises the following steps：
(1) using the yeast extract powder peptone glucose culture solution of embodiment 3, temperature be 32 DEG C, rotating speed be 250rpm, training
It is 18h to support the yeast cells time；Separation, collects yeast cells；
(2) yeast cells is washed with the lavation buffer solution of embodiment 65 times after collecting cell, and is delayed with the broken of embodiment 9
Fliud flushing is resuspended, and is crushed with sonioation method, is that 30000 × g is centrifuged in rotating speed, supernatant is for acellular
The cell extract of protein synthesis.
Breaking method in the present embodiment can also use glass bead method.
The yeast extract synthesized for cell-free protein prepared by embodiment 10, embodiment 11, embodiment 12 is shown in figure
1。
Embodiment 13 is used for the cell extract that cell-free protein is synthesized, the purposes of external Fast back-projection algorithm protein.
It is used for yeast extract and additional reactant and the gene matter that cell-free protein is synthesized by prepared by embodiment 10
Grain mixing, is placed in isothermal reaction container, and 6h is placed at 30 DEG C.
The volume ratio of the cell extract, additional reactant and the gene plasmid that are synthesized for cell-free protein is 15:25:
1。
Additional reactant is by being 5 by volume:25:3:1 amino acid mixed liquor, reaction buffer, energy-supplemented liquid and
Concentration constitutes for the 50 μ g/ml ribonucleic acid polymerase aqueous solution：
Amino acid mixed liquor, solvent is deionized water；The concentration of every kind of amino acid is 10mmol/L equimolar concentration,
The amino acid includes：Glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, first
Methyllanthionine, tyrosine, serine, threonine, cysteine, asparagine, glutamine, aspartic acid, glutamic acid, bad ammonia
Acid, arginine, histidine；
Reaction buffer includes：55mmol/L 4- hydroxyethyl piperazineethanesulfonic acids, 100mmol/L potassium glutamates, 15mmol/L
Psicosoma, 25mmol/L 3-phoshoglyceric acids, 7.5mmol/L CAMPs, the core of 3.3mmol/L nicotinamide adenines two
Thuja acid, 3.3mmol/L coacetylases, 0.5mmol/L folinic acid, 3.3mg/mL transfer RNA (tRNA)s, 0.5mmol/L spermidines；Solvent
It is deionized water；
Energy-supplemented liquid includes：2mmol/L uridine triphosphates, 2mmol/L cytidines, 2mmol/L atriphos,
6mmol/L GTPs, solvent is deionized water.
Gene plasmid is that pRset-eGFP (is purchased from：Promega Corporation).
Embodiment 14
It is used for yeast extract and additional reactant and the gene matter that cell-free protein is synthesized by prepared by embodiment 11
Grain mixing, is placed in isothermal reaction container, and 6h is placed at 30 DEG C.
The volume ratio of the cell extract, additional reactant and the gene plasmid that are synthesized for cell-free protein is 15:25:
1
Additional reactant is by being 5 by volume:25:3:1 amino acid mixed liquor, reaction buffer, energy-supplemented liquid and
Concentration constitutes for the 50 μ g/ml ribonucleic acid polymerase aqueous solution：
Amino acid mixed liquor, solvent is deionized water；The concentration of every kind of amino acid is 10mmol/L equimolar concentration,
The amino acid includes：Glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, first
Methyllanthionine, tyrosine, serine, threonine, cysteine, asparagine, glutamine, aspartic acid, glutamic acid, bad ammonia
Acid, arginine, histidine；
Reaction buffer includes：55mmol/L 4- hydroxyethyl piperazineethanesulfonic acids, 100mmol/L potassium glutamates, 15mmol/L
Psicosoma, 25mmol/L 3-phoshoglyceric acids, 7.5mmol/L CAMPs, the core of 3.3mmol/L nicotinamide adenines two
Thuja acid, 3.3mmol/L coacetylases, 0.5mmol/L folinic acid, 3.3mg/mL transfer RNA (tRNA)s, 0.5mmol/L spermidines；Solvent
It is deionized water；
Energy-supplemented liquid includes：2mmol/L uridine triphosphates, 2mmol/L cytidines, 2mmol/L atriphos,
6mmol/L GTPs, solvent is deionized water.
Gene plasmid is that pRset-eGFP (is purchased from：Promega Corporation).
Embodiment 15
It is used for yeast extract and additional reactant and the gene matter that cell-free protein is synthesized by prepared by embodiment 12
Grain mixing, is placed in isothermal reaction container, and 6h is placed at 30 DEG C.
The volume ratio of the cell extract, additional reactant and the gene plasmid that are synthesized for cell-free protein is 15:25:
1
Additional reactant is by being 5 by volume:25:3:1 amino acid mixed liquor, reaction buffer, energy-supplemented liquid and
Concentration constitutes for the 50 μ g/ml ribonucleic acid polymerase aqueous solution：
Amino acid mixed liquor, solvent is deionized water；The concentration of every kind of amino acid is 10mmol/L equimolar concentration,
The amino acid includes：Glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, first
Methyllanthionine, tyrosine, serine, threonine, cysteine, asparagine, glutamine, aspartic acid, glutamic acid, bad ammonia
Acid, arginine, histidine；
Reaction buffer includes：55mmol/L 4- hydroxyethyl piperazineethanesulfonic acids, 100mmol/L potassium glutamates, 15mmol/L
Psicosoma, 25mmol/L 3-phoshoglyceric acids, 7.5mmol/L CAMPs, the core of 3.3mmol/L nicotinamide adenines two
Thuja acid, 3.3mmol/L coacetylases, 0.5mmol/L folinic acid, 3.3mg/mL transfer RNA (tRNA)s, 0.5mmol/L spermidines；Solvent
It is deionized water；
Energy-supplemented liquid includes：2mmol/L uridine triphosphates, 2mmol/L cytidines, 2mmol/L atriphos,
6mmol/L GTPs, solvent is deionized water.
Gene plasmid is that pRset-eGFP (is purchased from：Promega Corporation).
The protein output that embodiment 13, embodiment 14, embodiment 15 are synthesized is shown in Fig. 2.
It is demonstrated experimentally that other yeast cells can be used for the present invention.
Claims (9)
1. a kind of preparation method of the cell extract synthesized for cell-free protein, it is characterised in that comprise the following steps：
(1) yeast extract powder peptone glucose culture solution culture yeasts cell is used, yeast cells is collected in separation；
(2) yeast cells collected is washed 2-5 times using lavation buffer solution, and is suspended again with disruption buffer, is crushed；
Centrifuge, supernatant is to be used for the cell extract that cell-free protein is synthesized.
2. according to the method described in claim 1, it is characterised in that the composition of the yeast extract powder peptone glucose culture solution：
10-30g/L yeast extracts, 5-15g/L peptones, 25-35g/L glucose, solvent is deionized water.
3. according to the method described in claim 1, it is characterised in that the condition of the culture yeasts cell is：25 DEG C -32 of temperature
DEG C, rotating speed be 150rpm-250rpm, the time is 3-18h.
4. according to the method described in claim 1, it is characterised in that the composition of lavation buffer solution：PH=7.2-7.6 20-
50mmol/L4- hydroxyethyl piperazineethanesulfonic acids-potassium hydroxide, 80-120mmol/L potassium glutamates, 1-4mmol/L psicosomas, 1-
4mmol/L dithiothreitol (DTT)s, 50g/L-100g/L mannitol；Solvent is deionized water.
5. according to the method described in claim 1, it is characterised in that the composition of disruption buffer：PH=7.2-7.6 20-
50mmol/L 4- hydroxyethyl piperazineethanesulfonic acids-potassium hydroxide, 80-120mmol/L potassium glutamates, 1-4mmol/L psicosomas,
1-4mmol/L dithiothreitol (DTT)s, 50g/L-100g/L mannitol, 0.2-0.8mmol/L phenylmethylsulfonyl fluorides；Solvent is deionization
Water.
6. according to the method described in claim 1, it is characterised in that step (2) the broken method is Mechanical Method, high pressure is even
Slurry processes, sonioation method or glass bead method.
7. according to the method described in claim 1, it is characterised in that the rotating speed of step (2) described centrifugation is 20000 × g-30000
×g。
8. prepared by the method described in one of claim 1-7 is used for the cell extract that cell-free protein is synthesized.
9. the cell extract for being used for cell-free protein synthesis of claim 8 is used for the use of external Fast back-projection algorithm protein
On the way.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551745A (en) * | 2018-05-31 | 2019-12-10 | 康码（上海）生物科技有限公司 | Multiple histidine sequence tag and application thereof in protein expression and purification |
| CN111996206A (en) * | 2020-06-19 | 2020-11-27 | 清华大学 | Light-operated cell-free protein synthesis method, plasmid used by method and product using method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1530373A (en) * | 2003-01-07 | 2004-09-22 | ��ʽ���絺���������� | Yeast extracted liquid for cell-free protein synthesis, preparation thereof, and cell-free protein synthesis therewith |
| WO2014144583A2 (en) * | 2013-03-15 | 2014-09-18 | Northwestern University | Methods for cell- free protein synthesis |
| WO2015193897A1 (en) * | 2014-06-17 | 2015-12-23 | B G Negev Technologies And Applications Ltd At Ben | Genetically expanded cell free protein synthesis systems, methods and kits |
-
2017
- 2017-07-21 CN CN201710599126.9A patent/CN107299058A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1530373A (en) * | 2003-01-07 | 2004-09-22 | ��ʽ���絺���������� | Yeast extracted liquid for cell-free protein synthesis, preparation thereof, and cell-free protein synthesis therewith |
| WO2014144583A2 (en) * | 2013-03-15 | 2014-09-18 | Northwestern University | Methods for cell- free protein synthesis |
| WO2015193897A1 (en) * | 2014-06-17 | 2015-12-23 | B G Negev Technologies And Applications Ltd At Ben | Genetically expanded cell free protein synthesis systems, methods and kits |
Non-Patent Citations (1)
| Title |
|---|
| 王雪等: "不同细胞破碎方法对无细胞蛋白表达系统细胞抽提物活性的影响", 《中国生物工程杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551745A (en) * | 2018-05-31 | 2019-12-10 | 康码（上海）生物科技有限公司 | Multiple histidine sequence tag and application thereof in protein expression and purification |
| CN111996206A (en) * | 2020-06-19 | 2020-11-27 | 清华大学 | Light-operated cell-free protein synthesis method, plasmid used by method and product using method |
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Address after: 300350 District, Jinnan District, Tianjin Haihe Education Park, 135 beautiful road, Beiyang campus of Tianjin University Applicant after: Tianjin University Address before: 300072 Tianjin City, Nankai District Wei Jin Road No. 92 Applicant before: Tianjin University |
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