Cell-free translation system, method and product
阅读说明：本技术 一种无细胞翻译体系、方法及产物 (Cell-free translation system, method and product ) 是由 张晓荣 赵谦 于 2021-07-07 设计创作，主要内容包括：本文涉及技术属于分子生物学领域,尤其涉及一种无细胞翻译体系、方法及产物。所述翻译体系包括：5’帽子及3’多聚腺苷酸的mRNA、氨基酸混合物、能量分子、mRNA翻译酶源的混合物；所述能量分子包括磷酸肌酸、ATP和鸟苷三磷酸(GTP)。所述翻译体系经过mRNA转录、体外转录和凝胶电泳分析确定可以迅速获得大量目的蛋白并具有以下特点：采用细胞外翻译技术克服了原核细胞表达部分基因不能表达问题；采用细胞外翻译技术克服了真核细胞表达周期长效率低问题；采用添加磷酸肌酸等能量小分子的策略优化了原有细胞外翻译技术的活性低、效率差问题；采用5’帽子及3’多聚腺苷酸的mRNA增强稳定性和与受体结合速度及整体翻译速度。(The present disclosure relates to the field of molecular biology, and more particularly to a cell-free translation system, method, and product. The translation system comprises: a mixture of a 5 'cap and a 3' poly A mRNA, a mixture of amino acids, an energy molecule, a source of mRNA translation enzyme; the energy molecules include phosphocreatine, ATP, and Guanosine Triphosphate (GTP). The translation system is determined by mRNA transcription, in vitro transcription and gel electrophoresis analysis to be capable of rapidly obtaining a large amount of target protein and has the following characteristics: the problem that part of genes expressed by prokaryotic cells cannot be expressed is solved by adopting an extracellular translation technology; the problem of long expression period and low efficiency of eukaryotic cells is solved by adopting an extracellular translation technology; the strategy of adding energy micromolecules such as creatine phosphate and the like is adopted to optimize the problems of low activity and poor efficiency of the original extracellular translation technology; the use of 5 'caps and 3' poly A mRNA enhances stability and receptor binding rates and overall translation rates.)
1. A cell-free translation system, comprising: a mixture of a 5 'cap and a 3' poly A mRNA, a mixture of amino acids, an energy molecule, and a source of mRNA translation enzyme; the energy molecule comprises phosphocreatine.
2. The translation system of claim 1, wherein said energy molecule further comprises ATP and Guanosine Triphosphate (GTP).
3. The translation system according to claim 1 or 2, wherein said mixture of amino acids comprises a mixture of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
4. The translation system of claim 1 or 2, wherein the source of mRNA translator comprises eukaryotic cell lysis supernatant.
5. A cell-free translation method comprising the translation system according to any one of claims 1 to 4, comprising the steps of:
transcribing mRNA having a 5 'cap and a 3' poly A;
adding the mRNA, the amino acid mixture, the energy molecule and the mRNA translation enzyme source into a reaction container, and reacting to obtain a mixed solution I containing the target protein;
and (3) processing the mixed solution I to obtain the target protein.
6. The translation method according to claim 5, wherein said energy molecule comprises ATP, phosphocreatine, and Guanosine Triphosphate (GTP).
7. The translation process of claim 5, wherein said source of mRNA translators comprises eukaryotic cell lysis supernatant, prepared by eukaryotic cell lysis centrifugation.
8. The translation method according to claim 5 or 6, wherein said reaction condition in said step (2) is a reaction temperature of 35-39 ℃ for 10-120 min.
9. The translation method according to claim 5 or 6, wherein said step (3) treatment method comprises gel electrophoresis analysis.
10. A cell-free translation product produced by the translation system according to any one of claims 1 to 4 and the translation method according to any one of claims 5 to 8.
Technical Field
The technology belongs to the field of molecular biology, and particularly relates to a cell-free translation system, a cell-free translation method and a cell-free translation product.
Background
The final product of gene expression mainly exists in the form of protein except non-coding RNA, and the existing protein expression technology mainly comprises prokaryotic bacterial expression, eukaryotic insect cell expression and humanized engineering cell expression. The inventor of the invention finds that: the main advantage of prokaryotic cell expression is low cost, but many proteins cannot be expressed in the prokaryotic cell; eukaryotic cells have high expression efficiency, but the problems of high cost and long time period exist at present; the existing extracellular translation technology has the problems of low activity, low efficiency, poor economy and the like.
Disclosure of Invention
The researchers of the invention find that the 5' end of the hat has the structural function through research:
(1) stabilizing the primary structure of the mRNA to prevent hydrolysis of the mRNA by 5' -exonuclease;
(2) providing a signal for mRNA recognition ribosome, so that mRNA can be quickly combined with ribosome, and the synthesis efficiency of mRNA to protein is improved;
function of 3' terminal polyA tail Structure
(1) Facilitates the transfer of mRNA from the nucleus to the cytoplasm;
(2) has an effect on the synthesis rate of the protein;
(3) in connection with the protection of the stability of the mRNA and the maintenance of the secondary structure of the mRNA.
Further, the researchers of the present invention found through research that eukaryotic cell mRNA is translated in extracellular environment with low efficiency, and mainly due to enzyme system: mRNA translation requires a complex enzyme system, and the artificial combination of the enzyme system is difficult to completely fit the real system composition in the cell, so that the extracellular translation efficiency is restricted.
Further, the researchers of the present invention found through research that eukaryotic mRNA is translated in extracellular environment with low efficiency, and mainly limited by energy supply system: the reason why higher translation efficiency cannot be obtained by using conventional ATP in a holoenzyme system provided by using a eukaryotic cell lysate is that eukaryotic cell mRNA is translated and supplied with energy, unlike prokaryotes, energy supplying substances such as guanosine triphosphate and phosphocreatine are required in addition to ATP.
An object of an embodiment of the present invention is to provide a cell-free translation system including: 5 'cap and 3' poly A mRNA, amino acid mixture, energy molecule, mRNA translation enzyme source; the energy molecule comprises phosphocreatine.
The energy molecule also includes ATP and Guanosine Triphosphate (GTP).
Preferably, the source of mRNA translators comprises eukaryotic cell lysis supernatant; the amino acid mixture comprises a mixture of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
Preferably, the source of mRNA translators comprises eukaryotic cell lysis supernatant.
A cell-free translation method comprises the above translation system, and comprises the following steps:
transcribing mRNA having a 5 'cap and a 3' poly A;
adding the mRNA, the amino acid mixture, the energy molecule and the mRNA translation enzyme source into a reaction container, and reacting to obtain a mixed solution I containing the target protein;
and (3) processing the mixed solution I to obtain the target protein.
Preferably, the energy molecule comprises ATP, phosphocreatine, and Guanosine Triphosphate (GTP); preferably, the source of mRNA translators comprises eukaryotic cell lysis supernatant prepared by eukaryotic cell lysis centrifugation.
Preferably, the reaction condition in the step (2) is that the reaction is carried out at the temperature of 35-39 ℃ for 10-120 min.
Preferably, the processing method in step (3) comprises gel electrophoresis analysis.
Preferably, all the operations in the following examples should be performed at low temperature, and the operations other than the step (2) reaction should be performed on ice as much as possible.
A cell-free translation product, which is prepared by the translation system and the translation method.
The RNA reaction system comprises A, U, C, G ribonic acid monomers, ATP, the corresponding DNA duplex, and RNA polymerase.
Preferably, the necessary buffer is added.
The beneficial effects include:
the invention provides a cell-free translation system, a method and a product, which have the following characteristics: 1. the problem that part of genes expressed by prokaryotic cells cannot be expressed is solved by adopting an extracellular translation technology; 2. the problem of long expression period and low efficiency of eukaryotic cells is solved by adopting an extracellular translation technology; 3. the strategy of adding energy micromolecules such as creatine phosphate and the like is adopted to optimize the problems of low activity and poor efficiency of the original extracellular translation technology; 4. the use of 5 'caps and 3' poly A mRNA enhances stability and receptor binding rates and overall translation rates.
Drawings
FIG. 1 is a flow chart of a cell-free translation method;
FIG. 2 is an electrophoretic analysis chart of the cell-free translation product in example 5;
FIG. 3 is a graph showing the quantitative analysis of cell-free translation products in example 3;
FIG. 4 is a graph showing the quantitative analysis of cell-free translation products in comparative example 2;
FIG. 5 is a graph showing the quantitative analysis of cell-free translation products in comparative example 3.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the scope of the present invention.
A cell-free translation system comprising: a mixture of a 5 'cap and a 3' poly A mRNA, a mixture of amino acids, an energy molecule, and a source of mRNA translation enzyme; the energy molecule comprises phosphocreatine.
The energy molecule also includes ATP and Guanosine Triphosphate (GTP).
Preferably, the source of mRNA translators comprises eukaryotic cell lysis supernatant; a mixture comprising alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
A cell-free translation method comprises the above translation system, and comprises the following steps:
transcribing mRNA having a 5 'cap and a 3' poly A;
adding the mRNA, the amino acid mixture, the ATP and an mRNA translation enzyme source into a reaction container, and reacting to obtain a first mixed solution containing the target protein;
and (3) processing the mixed solution I to obtain the target protein.
Preferably, Guanosine Triphosphate (GTP) is added in the step (2); the mRNA translator source comprises eukaryotic cell lysis supernatant, and the eukaryotic cell lysis supernatant is prepared by eukaryotic cell lysis centrifugation.
Preferably, the reaction condition in the step (2) is that the reaction is carried out at the temperature of 35-39 ℃ for 10-120 min.
Preferably, the processing method in step (3) comprises gel electrophoresis analysis.
A cell-free translation product, which is prepared by the translation system and the translation method.
The RNA reaction system comprises A, U, C, G ribonic acid monomers, ATP, the corresponding DNA duplex, and RNA polymerase.
Preferably, the necessary buffer is added
In order to better illustrate the beneficial effects of the cell-free translation product, the embodiment also discloses a prokaryotic intracellular gene transfer technology, wherein the method is a retrovirus vector-mediated transfection method, and a phage virus is selected for prokaryotic cell gene transfer.
In order to better illustrate the beneficial effects of the cell-free translation product, the embodiment also discloses a eukaryotic nuclear cell gene transfer technology, wherein the method is a method for transferring genes in eukaryotic cells by taking retrovirus as a vector-mediated transfection method, and phage virus or corresponding animal and plant viruses are selected for gene transfer in eukaryotic cells.
In order to better illustrate the beneficial effects of the cell-free translation product, the embodiment also discloses a method for analyzing protein by electrophoresis, SDS-polyacrylamide gel electrophoresis, which is characterized in that SDS (sodium dodecyl sulfate) is introduced into a polyacrylamide gel system, the SDS can break intramolecular and intermolecular hydrogen bonds to destroy the secondary and tertiary structures of the protein, a strong reducing agent can break disulfide bonds between cysteine, the protein is combined with SDS molecules in a certain concentration of SDS solution containing the strong reducing agent according to a certain proportion to form an SDS-protein complex with negative charges, and the complex combines a large amount of SDS to ensure that the protein loses the original charge state to form a negative ion block which only keeps the original molecular size as a characteristic, thereby reducing or eliminating the natural charge difference between various protein molecules, and the combination of the SDS and the protein is in proportion by weight, thus, the migration speed of protein molecules when subjected to electrophoresis depends on the size of the molecules. One of the key points for the success of SDS electrophoresis is the degree of protein binding to SDS during the electrophoresis, especially during the sample preparation, as follows:
(1) cleaning the glass plate with distilled water, and air-drying to prepare 2 clean conical flasks;
(2) when the glass plate is fixed on the glue pouring support, the force applied to the two sides must be uniform, so that the glass plate is prevented from being damaged;
(3) preparing separation gel according to a proportion, quickly adding the separation gel by a pipette, adding a little distilled water about 5 cm, and standing for 40 minutes;
(4) the gel preparation process needs to be rapid, the catalyst TEMED needs to be added before gel injection, otherwise, the gel cannot be injected by condensation, and the gel injection process is preferably finished at one time, so that bubbles are prevented from being generated;
(5) the purpose of water sealing is to make the separation gel straight and eliminate air bubbles, and the mark of good gel polymerization is that a clear interface is formed between the gel and a water layer;
(6) pouring out water, sucking the residual water by using filter paper, preparing concentrated glue according to a proportion, continuously and stably adding the concentrated glue to a position 5mm away from the edge, quickly inserting into a sample comb, and standing for 40 minutes;
(7) the sample comb needs to be inserted stably at one time, air bubbles cannot exist at the comb opening, and the comb bottom needs to be horizontal;
(8) after the sample comb is pulled out, adding a buffer solution into the upper groove, and removing agarose at the bottom end when the saw teeth are submerged;
(9) if the bubbles in the sawtooth hole are all discharged, otherwise, the sample adding effect is influenced;
(10) three samples are added, 1) 10 mul of standard protein dissolving solution is taken in an EP tube, 10 mul of 2 times sample buffer solution is added, and the sample loading amount is 20 mul; 2) taking 10 mul of the sample 1 solution, and then adding 10 mul of 2-time sample buffer solution, wherein the sample loading amount is 5 mul and 10 mul respectively;
(11) injecting sample from one third of the bottom of the tank by using a micro-injector, heating the sample in boiling water for 3 minutes before sample injection, and removing metastable state polymerization; the injector can not be too low to puncture the colloid, and can not be too high, so that the sample can be diffused when sinking;
(12) in order to avoid edge effect, a hole in the middle is preferably selected for sample injection, a buffer solution is added into an electrophoresis tank, a power supply is switched on, electrophoresis is carried out, the current is kept constant at 10mA, when the current is changed to 20mA after entering separation gel and bromophenol blue is 5mm away from the edge of the gel, the electrophoresis is stopped;
(13) stripping and dyeing the gel plate: after electrophoresis is finished, prying open the glass plate, marking the gel plate, placing the gel plate in a large culture dish, adding a staining solution, and staining for about 1 hour;
(14) decolorizing, rinsing the dyed gel plate with distilled water for several times, and decolorizing with decolorizing solution until the protein zone is clear; the glue is carefully stripped, the glue is kept intact, and the dyeing is sufficient;
(15) and (5) analyzing an experimental result.
In some alternative embodiments, the problem that part of the gene expressed by the prokaryotic cell cannot be expressed is overcome by adopting an extracellular translation technology;
in some alternative embodiments, the problem of long expression period and low efficiency of eukaryotic cells is overcome by adopting an extracellular translation technology;
in some optional embodiments, the problems of low activity and poor efficiency of the original extracellular translation technology are optimized by adopting a strategy of adding energy micromolecules such as creatine phosphate and the like;
in some alternative embodiments, mRNA with 5 'caps and 3' polyadenylation regions are used to enhance stability and receptor binding rates and overall translation rates.
Example 1
Based on the disclosed embodiments, a cell-free translation system is disclosed, comprising: 5 'cap and 3' poly A mRNA, amino acid mixture, ATP and mRNA translation mixture.
The translation system further comprises Guanosine Triphosphate (GTP).
Preferably, the mRNA structure of the 5 'cap and 3' polyadenylation is:
preferably, the mRNA translation comprises eukaryotic cell lysis supernatant; the amino acid mixture is 20 disclosed amino acid mixtures.
The embodiment also discloses a cell-free translation method, which comprises the translation system and comprises the following steps:
transcribing mRNA having a 5 'cap and a 3' poly A;
adding the mRNA, the amino acid mixture, ATP, Guanosine Triphosphate (GTP) and mRNA into a reaction container, and reacting to obtain a first mixed solution containing the target protein;
and (3) processing the mixed solution I to obtain the target protein.
The mRNA translator source comprises eukaryotic cell lysis supernatant, and the eukaryotic cell lysis supernatant is prepared by eukaryotic cell lysis centrifugation.
The reaction condition of the step (2) is that the reaction is carried out for 10min at the temperature of 35 ℃.
The processing method in the step (3) comprises gel electrophoresis analysis.
The embodiment also discloses a cell-free translation product, which is prepared by the translation system and the translation method.
The RNA reaction system comprises A, U, C, G ribonic acid monomers, ATP, the corresponding DNA duplex, and RNA polymerase.
Preferably, the necessary buffer is added.
In some alternative embodiments, the problem that part of the gene expressed by the prokaryotic cell cannot be expressed is overcome by adopting an extracellular translation technology;
in some alternative embodiments, the problem of long expression period and low efficiency of eukaryotic cells is overcome by adopting an extracellular translation technology;
in some optional embodiments, the problems of low activity and poor efficiency of the original extracellular translation technology are optimized by adopting a strategy of adding energy micromolecules such as creatine phosphate and the like;
in some alternative embodiments, mRNA with 5 'caps and 3' polyadenylation regions are used to enhance stability and receptor binding rates and overall translation rates.
Example 2
Based on the disclosed embodiments, a cell-free translation system is disclosed, comprising: a mixture of 5 'cap and 3' polyadenylated mRNA, amino acid mixture, ATP and mRNA translation enzyme source.
The translation system further comprises Guanosine Triphosphate (GTP).
Preferably, the mRNA structure of the 5 'cap and 3' polyadenylation is:
preferably, the source of mRNA translators comprises eukaryotic cell lysis supernatant; the amino acid mixture is 20 disclosed amino acid mixtures.
The embodiment also discloses a cell-free translation method, which comprises the translation system and comprises the following steps:
transcribing mRNA having a 5 'cap and a 3' poly A;
adding the mRNA, the amino acid mixture, ATP, Guanosine Triphosphate (GTP) and an mRNA translation enzyme source into a reaction container, and reacting to obtain a first mixed solution containing the target protein;
and (3) processing the mixed solution I to obtain the target protein.
The mRNA translator source comprises eukaryotic cell lysis supernatant, and the eukaryotic cell lysis supernatant is prepared by eukaryotic cell lysis centrifugation.
The reaction condition of the step (2) is that the temperature is 39 ℃ and the reaction time is 120 min.
The processing method in the step (3) comprises gel electrophoresis analysis.
The embodiment also discloses a cell-free translation product, which is prepared by the translation system and the translation method.
The RNA reaction system comprises A, U, C, G ribonic acid monomers, ATP, the corresponding DNA duplex, and RNA polymerase.
Preferably, the necessary buffer is added.
In some alternative embodiments, the problem that part of the gene expressed by the prokaryotic cell cannot be expressed is overcome by adopting an extracellular translation technology;
in some alternative embodiments, the problem of long expression period and low efficiency of eukaryotic cells is overcome by adopting an extracellular translation technology;
in some optional embodiments, the problems of low activity and poor efficiency of the original extracellular translation technology are optimized by adopting a strategy of adding energy micromolecules such as creatine phosphate and the like;
in some alternative embodiments, mRNA with 5 'caps and 3' polyadenylation regions are used to enhance stability and receptor binding rates and overall translation rates.
Example 3
Based on the disclosed embodiments, a cell-free translation system is disclosed, comprising: a mixture of 5 'cap and 3' polyadenylated mRNA, amino acid mixture, ATP and mRNA translation enzyme source.
The translation system further comprises Guanosine Triphosphate (GTP).
Preferably, the mRNA structure of the 5 'cap and 3' polyadenylation is:
preferably, the source of mRNA translators comprises eukaryotic cell lysis supernatant; the amino acid mixture is 20 amino acid mixtures.
The embodiment also discloses a cell-free translation method, which comprises the translation system and comprises the following steps:
transcribing mRNA having a 5 'cap and a 3' poly A;
adding the mRNA, the amino acid mixture, ATP, Guanosine Triphosphate (GTP) and an mRNA translation enzyme source into a reaction container, and reacting to obtain a first mixed solution containing the target protein;
and (3) processing the mixed solution I to obtain the target protein.
The mRNA translator source comprises eukaryotic cell lysis supernatant, and the eukaryotic cell lysis supernatant is prepared by eukaryotic cell lysis centrifugation.
The reaction condition of the step (2) is that the temperature is 37 ℃ and the reaction time is 30 min.
The processing method in the step (3) comprises gel electrophoresis analysis.
The embodiment also discloses a cell-free translation product, which is prepared by the translation system and the translation method.
The RNA reaction system comprises A, U, C, G ribonic acid monomers, ATP, the corresponding DNA duplex, and RNA polymerase.
Preferably, the necessary buffer is added.
In some alternative embodiments, the problem that part of the gene expressed by the prokaryotic cell cannot be expressed is overcome by adopting an extracellular translation technology;
in some alternative embodiments, the problem of long expression period and low efficiency of eukaryotic cells is overcome by adopting an extracellular translation technology;
in some optional embodiments, the problems of low activity and poor efficiency of the original extracellular translation technology are optimized by adopting a strategy of adding energy micromolecules such as creatine phosphate and the like;
in some alternative embodiments, mRNA with 5 'caps and 3' polyadenylation regions are used to enhance stability and receptor binding rates and overall translation rates.
Example 4
On the basis of the disclosed examples, an example of in vitro preparation of mRNA containing cap structures (GFP mRNA is used in this example) was prepared using the T7 transcriptase system in the following reaction system:
t7 RNA transcription reaction system
Reacting A, U, C, G ribonic acid monomer, DNA double strand at the tail end 46-58A of the front end T7 promoter region and T7 RNA polymerase at 37 ℃ for 30 minutes to obtain target mRNA;
preparation of cell lysate
Selecting a large amount of commonly cultured cells, centrifuging 500g of cells for 5min, collecting, then cracking the cells, centrifuging and collecting supernatant;
synthesis of protein of interest
Adding target mRNA (GFP mRNA) into the lysate, adding necessary buffer solution, ATP and amino acids,
reacting for 1 hour at 37 ℃ to obtain target protein;
analysis of protein of interest
After the reaction is finished, the macroscopic green color can be seen in the centrifuge tube; the protein of interest was analyzed by gel electrophoresis as shown in FIG. 3.
Example 5
On the basis of the disclosed embodiments, an embodiment of in vitro preparation of the firefly luciferase with the cap structure is disclosed, and the specific preparation method is shown in example 3. As shown in FIG. 2, the results of the electrophoretic analysis showed that a large amount of the target protein was color-developed.
Example 6
On the basis of the disclosed embodiment, an embodiment for in vitro preparation of green fluorescent protein containing a cap structure is disclosed, and the specific preparation method is shown in example 3.
Comparative example 1
On the basis of the disclosed embodiment, an embodiment of preparing the firefly luciferase by culturing prokaryotic cells is disclosed, and the specific operation is to transfer the corresponding gene of the firefly luciferase into Escherichia coli prokaryotic cells and culture and separate the target protein according to the disclosed method.
The test results show that: the corresponding gene of the target protein cannot be expressed in the prokaryotic cell, and the target protein is hardly collected by adopting a corresponding analysis means of electrophoretic analysis, because the corresponding gene cannot be expressed in the prokaryotic cell due to the difference of expression modes of the prokaryotic cell and the eukaryotic cell.
Comparative example 2
On the basis of the disclosed embodiments, the embodiment of preparing the firefly luciferase by the eukaryotic cell culture is disclosed, and the operation is specifically to transfer the corresponding gene of the firefly luciferase into the eukaryotic cell and culture and separate the target protein according to the disclosed method.
The test results show that: as shown in FIG. 3/4, the gene corresponding to the target protein was only 10 in 30min after culturing in eukaryotic cells2Concentration of the target protein of up to 10% using a cell-free translation technique disclosed herein7And (4) concentration.
Comparative example 3
On the basis of the disclosed embodiment, the embodiment of preparing the firefly luciferase by the extracellular translation of mRNA is disclosed, and the specific operation is to culture the mRNA of the firefly luciferase corresponding to the gene extracellularly without adding energy molecule phosphocreatine and separate the target protein.
The test results show that: as shown in FIG. 3/5, the gene corresponding to the target protein only obtained 12-14 concentration within 30min when cultured in eukaryotic cells, whereas the target protein could reach 10 by using a cell-free translation technique disclosed herein7And (4) concentration.