CN111378708B

CN111378708B - In-vitro cell-free protein synthesis system and application thereof

Info

Publication number: CN111378708B
Application number: CN201811619819.0A
Authority: CN
Inventors: 郭敏; 徐开; 杨宁; 章小铃; 周子鉴; 于雪
Original assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Current assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2022-07-19
Anticipated expiration: 2038-12-28
Also published as: CN111378708A; WO2020135747A1

Abstract

The invention discloses an optimized in-vitro acellular protein synthesis system, which comprises the following components: a cell extract which is a yeast cell extract having an inserted T7 RNA polymerase gene; a saccharide substance which is a mixture of glucose and maltodextrin; a phosphoric acid compound; a buffer, said buffer being tris buffer; the DNA molecular template of the coding foreign protein is prepared by adopting a nucleic acid isothermal amplification method, and a sequence shown in SEQ ID NO.1 is inserted into the upstream of the coding sequence of the foreign protein in the DNA molecular template. Through optimization, the cost of in vitro protein synthesis is reduced, and the yield of the target protein is increased.

Description

In-vitro cell-free protein synthesis system and application thereof

Technical Field

The invention belongs to the technical field of protein synthesis, and particularly relates to a cell-free protein synthesis system for in vitro protein synthesis.

Background

Proteins are important molecules in cells, and are involved in performing almost all functions of cells. The difference in the sequence and structure of proteins determines their function (1). Within the cell, proteins can catalyze various biochemical reactions as enzymes, can coordinate various activities of the organism as signaling molecules, can support biological morphology, store energy, transport molecules, and mobilize the organism (2). In the biomedical field, protein antibodies are important means for treating diseases such as cancer as targeted drugs (1, 2).

The conventional protein expression system refers to a molecular biology technique for expressing foreign genes by model organisms such as bacteria, fungi, plant cells or animal cells (3). With the development of science and technology, cell-free expression systems, also called in vitro protein synthesis systems, have come into play, which take exogenous target mRNA or DNA as a protein synthesis template, and can realize the synthesis of target proteins by artificially controlling and supplementing substrates required by protein synthesis and substances such as transcription and translation related protein factors and the like (3, 4). The expression of proteins in vitro translation systems does not require the steps of plasmid construction, transformation, cell culture, cell collection and disruption, and is a rapid, time-saving and convenient protein expression mode (5, 6). The in vitro protein synthesis system is generally characterized in that components such as mRNA or DNA template, RNA polymerase, amino acid, ATP and the like are added into a lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of foreign proteins (5, 7).

Currently, commercial in vitro protein expression systems that are frequently tested include the e.coli system (ECE), Rabbit Reticulocyte Lysate (RRL), Wheat Germ (WGE), Insect Cell Extract (ICE) and human-derived systems (5, 6). Compared with the traditional in vivo recombinant expression system, the in vitro cell-free protein synthesis system has multiple advantages, such as the capability of expressing special proteins which have toxic action on cells or contain unnatural amino acids (such as D-amino acids), capability of simultaneously synthesizing multiple proteins in parallel by directly taking plasmids or PCR products as templates, and development of high-throughput drug screening and proteomics research (7). Commercially, E.coli in vitro synthesis systems are widely used. The Escherichia coli is easy to culture and ferment, has low cost and simple broken cells, and can synthesize protein (6) with high yield. Compared with prokaryotic systems, eukaryotic cells have high difficulty and high cost in culture, and the preparation process of cell extracts is complicated, so that translation systems of the eukaryotic cells have high cost and are only suitable for special laboratories (1, 2). Therefore, eukaryotic in vitro protein expression systems suitable for industrial large-scale (ton-scale) preparation and production do not exist at present.

After a stage of research and development and preparation, a high-yield and low-cost in vitro expression system, namely a yeast extract prepared by a high-pressure crushing method or a liquid nitrogen crushing method and added with magnesium acetate, potassium acetate, amino acid, ATP, a DNA template, polyethylene glycol and the like, has been developed in the field. However, the bulk exoprotein synthesis reaction system still has the disadvantages of high cost and low reaction efficiency, so that the establishment of a low-cost and high-efficiency in-vitro protein synthesis reaction system is urgently needed.

1. Garcia RA, Riley MR. Applied biochemistry and biotechnology. Humana Press,; 1981. 263-264 p.

2. Fromm HJ, Hargrove M. Essentials of Biochemistry. 2012;

3. Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, et al. Protein production and purification. Nat Methods. 2008;5(2):135–46.

4. Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol [Internet]. 2013;23(3):393–402. Available from: http://dx.doi.org/10.1016/j.sbi.2013.03.008

5. Katzen F, Chang G, Kudlicki W. The past, present and future of cell-free protein synthesis. Trends Biotechnol. 2005;23(3):150–6.

6. Lu Y. Cell-free synthetic biology: Engineering in an open world. Synth Syst Biotechnol [Internet]. 2017;2(1):23–7. Available from: http://linkinghub.elsevier.com/retrieve/pii/S2405805X1730008X

7. Spirin AS, Swartz JR. Chapter 1. Cell-Free Protein Synthesis Systems: Historical Landmarks, Classification, and General Methods. Wiley‐VCH Verlag GmbH & Co. KGaA; 2008. 1-34 p。

Disclosure of Invention

The invention aims to provide a low-cost and high-efficiency in-vitro protein synthesis reaction system. Mainly solves the technical problems of overhigh cost and low reaction efficiency of a protein synthesis system in the prior art.

The technical scheme adopted by the invention for solving the technical problems is as follows:

an in vitro cell-free protein synthesis system, said cell-free protein synthesis system comprising:

(a) a cell extract which is a yeast cell extract having an inserted T7 RNA polymerase gene;

(b) a saccharide substance which is a mixture of glucose and maltodextrin;

(c) a phosphoric acid compound;

(d) a buffer, said buffer being tris buffer;

(e) the DNA molecular template of the coding foreign protein is prepared by adopting a nucleic acid isothermal amplification method, and a sequence shown in SEQ ID NO.1 is inserted into the upstream of the coding sequence of the foreign protein in the DNA molecular template.

Preferably, the protein synthesis system further comprises one or more components of the group consisting of:

(f1) polyethylene glycol;

(f2) a substrate for RNA synthesis;

(f3) a mixture of amino acids;

(f4) magnesium ions;

(f5) potassium ions;

(f6) dithiothreitol (DTT);

(f7) an active enzyme capable of catalyzing the metabolism of a carbohydrate to produce ATP;

(f8) optionally water or an aqueous solvent.

Further preferably, the active enzyme is selected from amylase, phosphorylase, galactosidase, phosphoglucomutase, or a combination thereof. Further preferably, the amylase is an alpha amylase.

Preferably, the phosphate compound is selected from orthophosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or a combination thereof.

Preferably, the cell extract is derived from one or more types of cells selected from the group consisting of: escherichia coli, bacteria, mammalian cells, plant cells, yeast cells, or a combination thereof; preferably, the yeast cell is selected from saccharomyces cerevisiae, pichia pastoris, kluyveromyces, or a combination thereof; more preferably, the kluyveromyces lactis is kluyveromyces lactis.

Preferably, the concentration of the glucose is 8.8-128 mmol/L.

Preferably, the concentration of the maltodextrin is 84-500 mmol/L.

Preferably, the concentration (v/v) of the cell extract is 20% to 70%, preferably 30% to 60%, more preferably 40% to 50%, based on the total volume of the protein synthesis system.

The invention also provides a kit comprising a container and the above cell-free protein synthesis system components located in the container.

The invention also provides a method for synthesizing the in vitro exogenous protein, which comprises the following steps:

(i) providing the above in vitro cell-free protein synthesis system;

(ii) performing an incubation reaction under suitable conditions to synthesize the foreign protein.

Preferably, the method further comprises: (iii) optionally isolating or detecting said foreign protein from said in vitro cell-free protein synthesis system.

Compared with the prior art, the invention has the following beneficial effects:

1, replacing compounds containing high-energy phosphate bonds such as phosphoenolpyruvate, creatine phosphate and acetyl phosphate with low-cost substances such as glucose and maltodextrin, serving as energy sources to provide ATP for in vitro reaction, reducing cost and prolonging reaction time and increasing the yield of target protein in a slow-release energy providing mode.

2, the reaction efficiency is improved by changing the type, concentration and pH of the reaction buffer solution to provide a stable reaction environment for the in vitro protein synthesis reaction system. The yeast genome is modified, so that the additional expensive biological enzyme (such as RNA polymerase) is not needed in the reaction system. The mixed solution of the nucleoside triphosphate is prepared by using rNTP powder and buffer solution to replace the mixed solution of commercial rNTP on the market, so that the reaction cost is greatly reduced on the basis of not reducing the activity of an in vitro protein synthesis reaction system; the yield of the target in unit time is improved through the optimization of the DNA template; the DNA molecular template for coding the exogenous protein is prepared by utilizing the nucleic acid isothermal amplification technology, and the protein synthesis with high efficiency, high flux, simplicity and convenience is completed by utilizing the DNA template with extremely small amount (nanogram-microgram).

3, the invention can greatly reduce the cost of in vitro protein synthesis reaction, and simultaneously improve the in vitro protein synthesis capacity by more than 30 times.

Drawings

FIG. 1 is a graph showing the results of data on RFU values of fluorescent proteins synthesized by two protein synthesis systems in example 2 of the present invention; wherein the system 1 is the optimized protein synthesis system of the embodiment 2, the system 2 is the original protein synthesis system of the embodiment 2, the negative control is that no DNA template is added into the system 1, and the detection time is respectively 3 hours and 20 hours.

FIG. 2 is a graph showing the results of data on RFU values of fluorescent proteins synthesized by the maltodextrin + glucoprotein synthesis system in example 1 of the present invention; wherein the glucose concentration is 20mM, the maltodextrin concentration is 0-500 mM, and the detection time is 3 hours and 20 hours respectively. NC is a protein synthesis system without the addition of DNA template.

FIG. 3 is a graph showing the result of data on RFU values of fluorescent proteins synthesized by the maltodextrin + glucoprotein synthesis system in example 1 of the present invention; wherein the glucose concentration is 0-200 mM, the maltodextrin concentration is 320 mM, and the detection time is 20 hours.

Detailed Description

In the present invention, the expressions "in vitro cell-free protein synthesis system", "in vitro expression system", "in vitro protein synthesis reaction system", "cell-free protein synthesis system", and the like have the same meanings.

The present inventors have extensively and intensively studied and found that in an in vitro protein synthesis system, creatine phosphate and creatine phosphate kinase are used as energy sources to provide ATP for in vitro reactions, and although ATP can be released through corresponding kinase reactions, a large amount of energy can be provided only at the beginning, rapidly and transiently, and these high-energy compounds have inhibitory effect on in vitro cell synthesis, cannot provide energy for a long time, and have high cost, which is not favorable for efficiency improvement and industrial application of in vitro protein synthesis systems.

Glucose, maltodextrin and a phosphate compound (potassium phosphate) are used as energy sources to carry out in-vitro biological reaction, ATP can be slowly released, the cost is reduced, and the method is a novel energy regeneration system capable of being industrialized. Through optimization, compared with a creatine phosphate and creatine phosphate kinase system, the RFU value of a reaction system containing glucose and maltodextrin is increased by more than 30 times; compared with a reaction system taking glucose as an energy source, the RFU value of the reaction system is increased by more than 5 times. Based on the method, the in vitro cell-free protein synthesis system is optimized from multiple aspects, and the high-efficiency and low-cost protein synthesis system is provided.

In vitro cell-free protein synthesis system:

in the present invention, the in vitro cell-free protein synthesis system is not particularly limited, and one preferred cell-free protein synthesis system is a yeast in vitro protein synthesis system, preferably a Kluyveromyces in vitro protein synthesis system (more preferably, a Kluyveromyces lactis in vitro protein synthesis system).

Yeast (yeast) combines the advantages of simple culture, efficient protein folding, and post-translational modification. Wherein, the Saccharomyces cerevisiae (Saccharomyces cerevisiae) and the Pichia pastoris (Pichia pastoris) are model organisms for expressing complex eukaryotic proteins and membrane proteins, and the yeast can also be used as a raw material for preparing an in vitro translation system.

Kluyveromyces (Kluyveromyces) is a species of ascosporogenous yeast, of which Kluyveromyces marxianus and Kluyveromyces lactis (Kluyveromyces lactis) are industrially widely used. In comparison with other yeasts, kluyveromyces lactis has many advantages such as superior secretion ability, better large-scale fermentation characteristics, a level of food safety, and the ability to modify proteins post-translationally.

The in vitro cell-free protein synthesis system of the invention comprises: (a) a cell extract which is a yeast cell extract having an inserted T7 RNA polymerase gene; (b) a saccharide substance which is a mixture of glucose and maltodextrin; (c) a phosphoric acid compound; (d) a buffer, said buffer being tris buffer; (e) the DNA molecular template of the coding foreign protein is prepared by adopting a nucleic acid isothermal amplification method, and a sequence shown in SEQ ID NO.1 is inserted into the upstream of the coding sequence of the foreign protein in the DNA molecular template.

Preferably, the protein synthesis system further comprises one or more components of the group consisting of: (f1) polyethylene glycol; (f2) a substrate for RNA synthesis; (f3) a mixture of amino acids; (f4) magnesium ions; (f5) potassium ions; (f6) dithiothreitol (DTT); (f7) an active enzyme capable of catalyzing the metabolism of a carbohydrate to produce ATP; (f8) optionally water or an aqueous solvent.

In the present invention, the content and purity of the cell extract are not particularly limited. Preferably, the concentration (v/v) of the cell extract is 20% to 70%, preferably 30% to 60%, more preferably 40% to 50%, based on the total volume of the protein synthesis system.

Further, the cell extract is an aqueous extract of yeast cells.

Further, the cell extract does not contain long-chain nucleic acid molecules endogenous to yeast.

Further, the substrate for synthesizing RNA comprises: one of nucleoside monophosphate, nucleoside triphosphate or a combination thereof.

Further, the amino acid mixture comprises: 20 natural amino acids and non-natural amino acids.

Further, the magnesium ions are derived from a magnesium ion source selected from the group consisting of: one or the combination of magnesium acetate and magnesium glutamate.

Further, the potassium ion is derived from a potassium ion source selected from the group consisting of: one or the combination of potassium acetate and potassium glutamate.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In the description of the embodiments, the concentrations of the nucleoside triphosphate mixture and the amino acid mixture refer to the concentration of a single substance in the mixture, not the total substance in the mixture.

Comparative example 1 in vitro cell-free protein Synthesis System containing creatine phosphate + creatine phosphate kinase

In vitro protein synthesis reaction system: 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes-KOH) at a final concentration of 22 mM, pH 7.4, 120 mM potassium acetate, 5.0 mM magnesium acetate, 1.5mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a concentration of 1.5 mM), 0.1mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, each at a concentration of 0.1 mM), 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg/mL creatine phosphate kinase, 0.03 mg/mL 7 RNA polymerase, 2% polyethylene glycol, 50% volume yeast cell extract, 15 ng/μ L enhanced green fluorescent protein DNA.

In vitro protein synthesis reaction: and mixing the reaction system uniformly and placing the mixture in an environment with the temperature of 20-30 ℃ for reaction.

Determination of fluorescent protein Activity: after the reaction, the reaction mixture was immediately placed in an Envision 2120 multifunctional microplate reader (Perkin Elmer), and the intensity of the Fluorescence signal was measured using a Relative Fluorescence Unit (RFU) as an activity Unit.

The relative light unit value RFU of the in vitro cell-free protein synthesis system is 60 under the condition of 20 ℃ for 3 hours. The yield of enhanced green fluorescent protein is 1.50 mug/mL. After 3 hours of reaction, the RFU value began to decline slowly.

Example 1 in vitro cell-free protein Synthesis System containing glucose + maltodextrin

In vitro protein synthesis reaction system: 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes-KOH) at a final concentration of 22 mM and pH 7.4, 120 mM potassium acetate, 5.0 mM magnesium acetate, 1.5mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate), 0.1mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 1.7 mM dithiothreitol, 20mM glucose, 20mM tripotassium phosphate, 0-500 mM maltodextrin (measured as glucose monomers), 0.002mg/mL alpha amylase, 0.03 mg/mL T7 RNA polymerase, 2% polyethylene glycol, 50% volume yeast cell extract, 15 ng/μ L enhanced green fluorescent protein DNA.

In vitro protein synthesis reaction: and mixing the reaction system uniformly and then placing the mixture in an environment with the temperature of 20-30 ℃ for reaction.

Fluorescent protein activity assay: immediately after the reaction, the reaction mixture was placed in an Envision 2120 multifunctional microplate reader (Perkin Elmer), and the intensity of the Fluorescence signal was measured using a Relative Fluorescence Unit (RFU) as an activity Unit.

FIG. 2 is a graph showing the result of data on RFU values of fluorescent proteins synthesized by the maltodextrin + glucoprotein synthesis system in this example; wherein the concentration of glucose is 20mM, the concentration of maltodextrin is 0-500 mM, the detection time is 3 hours and 20 hours respectively, and NC refers to a protein synthesis system without adding a DNA template. As can be seen from fig. 2: the highest yield of fluorescent protein was obtained when the concentration of maltodextrin was in the range of 256-400 mM. The relative light unit value RFU of the in vitro protein synthesis reaction system was about 1900 in the case of reaction at 20 ℃ for 20 hours with the addition of 320 mM maltodextrin. Enhanced green fluorescent protein production was 121.10. mu.g/mL.

The addition of alpha-amylase is favorable for hydrolysis of maltodextrin, and in this example, if the alpha-amylase is absent, the relative light unit value RFU of the in vitro protein synthesis reaction system is about 1750, and the yield of the enhanced green fluorescent protein is 111.06 mug/mL.

FIG. 3 is a graph showing the results of data on the RFU values of fluorescent protein obtained by fixing 320 mM of maltodextrin and testing the effect of glucose at various concentrations; wherein the glucose concentration is 0-200 mM, the maltodextrin concentration is 320 mM, and the detection time is 20 hours. As can be seen from fig. 3: the highest RFU value of the fluorescent protein is achieved when the glucose concentration is about 20 mM.

EXAMPLE 2 optimization of in vitro cell-free protein Synthesis System

2.1 original in vitro cell-free protein Synthesis System (System 2): 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes-KOH) at a final concentration of 22 mM, pH 7.4, 120 mM potassium acetate, 5mM magnesium acetate, 1.5mM commercial nucleoside triphosphate mixture liquid (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate), 0.1mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg/mL creatine phosphate kinase, 0.03 mg/mL T7 RNA polymerase, 2% polyethylene glycol, 50% by volume yeast cell extract, 15 ng/muL enhanced green fluorescent protein DNA (the DNA is prepared by adopting a traditional PCR method, and a sequence shown in SEQ ID NO.2 is inserted into the upstream of a sequence for coding green fluorescent protein in a DNA molecular template). The yeast cells used for the yeast cell extract in the synthesis system are not subjected to gene modification and cannot endogenously express RNA polymerase, so that T7 RNA polymerase needs to be added in an exogenous manner.

In vitro protein synthesis reaction: mixing the above materials, and reacting at 20-30 deg.C.

Determination of fluorescent protein Activity: immediately after the reaction, the reaction mixture was placed in an Envision 2120 multifunctional microplate reader (Perkin Elmer), and the intensity of the Fluorescence signal was measured using a Relative Fluorescence Unit (RFU) as an activity Unit.

2.2 optimized in vitro cell-free protein Synthesis System (System 1): Tris-HCl (Tris-HCl) at a final concentration of 22 mM, pH 8.0, potassium acetate 120 mM, magnesium acetate 5mM, four nucleoside triphosphates (prepared by mixing four nucleoside triphosphate powders with Tris-buffer pH 8.0), an amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine) at a final concentration of 1.5mM, 1.7 mM dithiothreitol, 20mM tripotassium phosphate, 20mM glucose, 320 mM maltodextrin (measured as glucose monomer), 0.002mg/mL alpha-amylase, 2% polyethylene glycol, 50% by volume of yeast cell extract, 15 ng/muL enhanced green fluorescent protein DNA (the DNA is prepared by a nucleic acid isothermal amplification method, and a sequence shown in SEQ ID NO.1 is inserted into the upstream of a coding sequence of a fluorescent protein in a DNA molecular template). The yeast cell used by the yeast cell extract in the synthesis system is inserted with a T7 RNA polymerase gene, so that the T7 RNA polymerase can be endogenously expressed without adding T7 RNA polymerase exogenously.

In vitro protein synthesis reaction: the above-mentioned system is uniformly mixed, placed in the environment of 20-30 deg.C and reacted.

The optimized in vitro cell-free protein synthesis system (system 1) has the following optimization means:

1, establishment of highly effective and stable buffer system

System buffer solution: because tris is one of buffering agents commonly used in biology, tris is widely used as a solvent for nucleic acids and proteins. Preferably, Tris-HCl buffer (pH 8.0) is used in place of 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes-KOH) buffer at pH 7.4.

2, nucleoside triphosphate powder substitution and buffer component optimization (high efficiency + low cost)

System 2: the 25 mM mixture of four nucleoside triphosphates, including Adenosine Triphosphate (ATP), Guanosine Triphosphate (GTP), cytosine nucleoside triphosphate (CTP) and Uridine Triphosphate (UTP), is a commercial nucleoside triphosphate mixture and is the highest component in unit cost among all reaction components.

Through optimization, a mixed solution of the nucleoside triphosphate is prepared by using the trihydroxymethyl aminomethane with the pH value of 8.0 and four kinds of nucleoside triphosphate powder, and the mixed solution replaces a directly purchased commercial nucleoside triphosphate mixed solution, so that a high RFU value is shown in an in-vitro protein synthesis system.

3, establishment of energy regeneration System

The phosphocreatine and phosphocreatine kinase are used as energy sources to provide ATP for in vitro reaction, and although ATP can be released through corresponding kinase reaction to generate energy, a large amount of energy can be provided only at the initial stage, rapidly and transiently, and the high-energy compounds have an inhibition effect on in vitro cell synthesis, cannot supply energy durably, are high in cost and are not beneficial to efficiency improvement and industrial application of in vitro protein synthesis systems.

Glucose, maltodextrin and phosphate compounds are used as energy sources to carry out in-vitro biological reaction, ATP can be slowly released, the cost is reduced, and the method is a novel energy regeneration system capable of being industrialized. Optimized, the use of 20mM final concentration of glucose, 320 mM maltodextrin and 20mM potassium phosphate as energy sources for in vitro protein synthesis systems showed higher RFU values in vitro protein synthesis systems.

Optimization of Yeast genome engineering

The system 2 needs to add RNA polymerase, and the RNA polymerase obtained by commercial and laboratory preparation methods has the problems of high cost and low purity. By modifying a yeast genome and adopting a yeast cell extract into which a T7 RNA polymerase gene is inserted (see the patent content of application No. 2017107685501 for the insertion method of the T7 RNA polymerase gene), the reaction system does not need to add expensive biological enzymes (such as RNA polymerase), and the cost of in vitro protein synthesis is reduced under the condition that the RFU value is not greatly changed.

5, integration of cell-free protein Synthesis by coupling DNA replication, transcription, translation

And amplifying a large amount of target protein DNA templates by using a PCR (polymerase chain reaction) mode. However, the PCR technology is dependent on temperature cycle, and usually requires higher temperature for denaturation of DNA template and amplification and extension of newly synthesized DNA molecule, while high temperature can cause denaturation and inactivation of protein factor in vitro synthesis system, so that it is not suitable for in vitro synthesis system. Compared with PCR technology, isothermal amplification of nucleic acids is characterized by the fact that amplification of nucleic acids is achieved under specific, relatively mild temperature conditions, thus enabling in vitro coupling of DNA replication, mRNA transcription and protein synthesis. Meanwhile, the DNA polymerases used for isothermal amplification of nucleic acid, including phi29DNA polymerase, T7 DNA polymerase and the like, have great advantages in temperature and amplification efficiency, so that a large amount of DNA molecules do not need to be prepared in advance, and the in-vitro synthesis of protein can be realized by only a small amount of DNA templates. Through optimization, the RFU values of the DNA replication, transcription and translation coupling system listed in the table 1 in the in vitro protein synthesis system reach the DNA template preparation mode of the original system for amplifying a large amount of target protein DNA templates by using a PCR mode.

TABLE 1

6, DNA template modification and optimization

In system 2: the 5' untranslated region has no IRES (KLNCE 102) and no GAA sequence, and the post-translational initiation site (ATG) has no fusion protein fragment and label, so that the translation initiation efficiency is low, and the aim of synthesizing the protein in vitro quickly, efficiently and at high flux can not be achieved.

After the DNA template in the system 1 is modified, a GAA sequence and an IRES (KLNCE 102) sequence are inserted in sequence from the 5 ' end to the 3 ' end before the T7 promoter and the omega sequence in the 5 ' untranslated region. The leader peptide sequence and the His tag sequence are inserted in sequence from the 5 'end to the 3' end of the target protein after the translation initiation site (ATG). Through optimization, the modified DNA template sequence is a sequence shown in SEQ ID NO.1 inserted into the upstream of the foreign protein coding sequence.

The optimized protein synthesis system (system 1) and the original protein synthesis system (system 2) are respectively placed in the environment of 20-30 ℃ for reaction.

Determination of fluorescent protein Activity: different fluorescence is observed during the reaction process, and the color gradually becomes darker in a certain period of time. After the reaction, the reaction mixture was immediately placed in an Envision 2120 multifunctional microplate reader (Perkin Elmer), read, selected with different filters, and the intensity of each Fluorescence signal was detected with a Relative Fluorescence Unit (RFU) as an activity Unit.

Referring to fig. 1, a graph of data results for RFU values for fluorescent proteins synthesized for two protein synthesis systems; wherein system 1 is an optimized protein synthesis system, system 2 is an original protein synthesis system, and the negative control is that no DNA template is added to system 1.

As can be seen from the data results of FIG. 1, the RFU value of the fluorescent protein synthesized by the optimized protein synthesis system is about 40 times that of the original protein synthesis system.

The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention and the scope of the invention is to be protected.

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Claims

1. An in vitro cell-free protein synthesis system, comprising:

(a) a cell extract;

(b) a saccharide substance which is a mixture of glucose and maltodextrin;

(c) a phosphoric acid compound; the phosphate compound is selected from orthophosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or a combination thereof;

(d) a buffering agent;

(e) a DNA molecular template for coding a foreign protein, wherein a sequence shown as SEQ ID NO.1 is inserted into the DNA molecular template at the upstream of a coding sequence of the foreign protein;

(f1) polyethylene glycol;

(f4) magnesium ions;

(f5) potassium ions;

(f6) dithiothreitol;

the cell extract is a Kluyveromyces cell extract, and the Kluyveromyces cell used by the Kluyveromyces cell extract is inserted with a T7 RNA polymerase gene or the Kluyveromyces cell extract is directly added with T7 RNA polymerase.

2. The in vitro cell-free protein synthesis system of claim 1, wherein the kluyveromyces is kluyveromyces lactis.

3. The in vitro cell-free protein synthesis system of claim 1, wherein the buffer is tris buffer.

4. The in vitro cell-free protein synthesis system of claim 3, wherein the buffer is tris buffer at pH 8.0.

5. The in vitro cell-free protein synthesis system of claim 4, wherein the buffer is Tris-HCl buffer at pH 8.0.

6. The in vitro cell-free protein synthesis system of claim 1, further comprising one or more components of the group consisting of:

(f2) a substrate for RNA synthesis;

(f3) a mixture of amino acids;

(f8) optionally water or an aqueous solvent.

7. The in vitro cell-free protein synthesis system of claim 6, wherein the active enzyme is an amylase, a galactosidase, a phosphorylase, a phosphoglucomutase, or a combination thereof.

8. The in vitro cell-free protein synthesis system of claim 7, wherein the amylase is an alpha amylase.

9. The in vitro cell-free protein synthesis system of claim 1, wherein the in vitro cell-free protein synthesis system further comprises a DNA polymerase.

10. The in vitro cell-free protein synthesis system of claim 9, wherein the DNA polymerase is used for isothermal amplification of nucleic acids.

11. The in vitro cell-free protein synthesis system of claim 9, wherein the DNA polymerase is phi29DNA polymerase, T7 DNA polymerase, or a combination thereof.

12. The in vitro cell-free protein synthesis system of claim 1, wherein the concentration of glucose is 8.8 to 128 mmol/L.

13. The in vitro cell-free protein synthesis system of claim 1, wherein the maltodextrin is present at a concentration of 84 to 500mmol/L as glucose monomers.

14. The in vitro cell-free protein synthesis system of claim 1, wherein the glucose is present at a concentration of 20 mM; the concentration of the maltodextrin is 22-500 mM, and the concentration of the maltodextrin is measured by glucose monomers.

15. The in vitro cell-free protein synthesis system of claim 1, wherein the glucose concentration is from 8.8 to 128 mM; the concentration of the maltodextrin was 320 mM, and the concentration of the maltodextrin was measured as glucose monomers.

16. The in vitro cell-free protein synthesis system of claim 1, wherein the glucose concentration is 20mM, the maltodextrin concentration is 320 mM, and the phosphate compound is 20mM potassium phosphate; wherein the concentration of said maltodextrin is measured as glucose monomer.

17. The in vitro cell-free protein synthesis system of claim 1, wherein the cellular extract has a volume specific concentration of 20% to 70% based on the total volume of the in vitro cell-free protein synthesis system.

18. The in vitro cell-free protein synthesis system of claim 17, wherein the cellular extract has a concentration of 30% to 60% by volume, based on the total volume of the in vitro cell-free protein synthesis system.

19. The in vitro cell-free protein synthesis system of claim 18, wherein the cellular extract has a volume specific concentration of 40% to 50% based on the total volume of the in vitro cell-free protein synthesis system.

20. The in vitro cell-free protein synthesis system of claim 1, wherein the in vitro cell-free protein synthesis system comprises a kluyveromyces cell extract, wherein the kluyveromyces cell extract employs a kluyveromyces cell with an inserted T7 RNA polymerase gene; the in vitro cell-free protein synthesis system further comprises the following components in final concentrations: 22 mM Tris-HCl pH 8.0, 120 mM potassium acetate, 5mM magnesium acetate, 1.5mM four nucleoside triphosphates, 0.1mM amino acid mixture, 1.7 mM dithiothreitol, 20mM tripotassium phosphate, 20mM glucose, 320 mM maltodextrin as glucose monomer, 0.002mg/mL alpha amylase, 2% polyethylene glycol, DNA molecular template encoding a foreign protein.

21. The in vitro cell-free protein synthesis system of claim 20, wherein the DNA molecule template encoding the exogenous protein is a DNA molecule template encoding an enhanced green fluorescent protein.

22. A kit comprising a container and components of the in vitro cell-free protein synthesis system of any one of claims 1-21 in the container.

23. A method for synthesizing a foreign protein in vitro, comprising:

(i) providing an in vitro cell-free protein synthesis system according to any one of claims 1 to 21;

24. The method for synthesizing an exogenous protein according to claim 23, wherein the DNA molecule template is prepared by isothermal nucleic acid amplification.

25. The method for synthesizing an exogenous protein according to claim 23, wherein the method further comprises: (iii) optionally isolating or detecting said foreign protein from said in vitro cell-free protein synthesis system.

26. The method for synthesizing an in vitro foreign protein according to claim 23, wherein the incubation reaction is performed at a temperature of 20 to 30 ℃.