CN111378707B - In-vitro cell-free protein synthesis system and application thereof - Google Patents
In-vitro cell-free protein synthesis system and application thereof Download PDFInfo
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Abstract
The invention discloses an in vitro cell-free protein synthesis system, which comprises: a cell extract; a saccharide which is maltodextrin, lactose, or a combination of maltodextrin and glucose, or a combination of lactose and glucose, or a combination of maltodextrin and lactose and glucose; a phosphoric acid compound. The invention utilizes low-cost substances such as glucose, maltodextrin, lactose and the like to replace energy sources such as phosphoenolpyruvic acid, creatine phosphate, acetyl phosphate and the like to provide ATP for in vitro reaction, reduces the cost, and prolongs the reaction time and increases the yield of target protein by a slow-release energy providing mode.
Description
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, the field has developed a high-yield and low-cost in vitro expression system, namely yeast extract prepared by a high-pressure crushing method or a liquid nitrogen crushing method is added with magnesium acetate, potassium acetate, amino acid, ATP, a DNA template, polyethylene glycol and other components. 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;
(b) a saccharide which is maltodextrin, lactose, or a combination of maltodextrin and glucose, or a combination of lactose and glucose, or a combination of maltodextrin and lactose and glucose;
(c) a phosphoric acid compound.
Preferably, the protein synthesis system further comprises an active enzyme capable of catalyzing the metabolism of carbohydrates to produce ATP.
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 protein synthesis system further comprises one or more components of the group consisting of:
(d1) polyethylene glycol;
(d2) a substrate for RNA synthesis;
(d3) a substrate for synthesizing a protein;
(d4) magnesium ions;
(d5) potassium ions;
(d6) a buffering agent;
(d7) an RNA polymerase;
(d8) dithiothreitol (DTT);
(d9) optionally a solvent, which is water or an aqueous solvent.
Preferably, the cell extract is derived from one or more types of cells selected from the group consisting of: escherichia coli, 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 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.
In the present invention, the content and purity of the cell extract are not particularly limited.
The invention also provides a kit, which comprises a container and the components of the cell-free protein synthesis system positioned in the container.
The invention also provides a method for synthesizing the in vitro exogenous protein, which comprises the following steps:
(i) providing an in vitro cell-free protein synthesis system as described above;
(ii) adding a DNA molecular template for encoding the foreign protein, and carrying out incubation reaction under a proper condition, thereby synthesizing 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 high-energy phosphate bond-containing compounds such as phosphoenolpyruvic acid, creatine phosphate and acetyl phosphate with low-cost substances such as glucose, maltodextrin, lactose or corresponding combinations thereof as energy sources to provide ATP for in vitro reaction, reducing cost and simultaneously prolonging reaction time and increasing yield of target protein in a slow-release energy providing manner.
2, the energy system of glucose and maltodextrin is utilized, so that the in vitro protein synthesis reaction cost can be greatly reduced, and simultaneously, the in vitro protein synthesis capacity is improved by more than 30 times compared with a phosphocreatine/phosphocreatine kinase system.
Drawings
FIG. 1 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. 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 0-200 mM, the maltodextrin concentration is 320 mM, and the detection time is 20 hours.
FIG. 3 is a graph showing the results of data on RFU values of fluorescent proteins synthesized by the individual glucoprotein synthesis system in comparative example 2 of the present invention; wherein the glucose concentration is 0-80 mM, and the detection time is 3 hours.
FIG. 4 is a graph showing the results of data on RFU values of fluorescent proteins synthesized by the single maltodextrin protein synthesis system in example 2 of the present invention; wherein the concentration of maltodextrin is 0-500 mM, and the detection time is 20 hours.
FIG. 5 is a graph showing the result of data on RFU values of fluorescent proteins synthesized by the lactose alone synthesis system in example 3 of the present invention; wherein the lactose concentration is 0-200 mM, and the detection time is 3 hours and 20 hours respectively.
FIG. 6 is a graph showing the results of data on RFU values of fluorescent proteins synthesized by the lactose + glucose protein synthesis system in example 4 of the present invention; wherein the lactose concentration is 0-400 mM, the glucose concentration is 20mM, and the detection time is 3 hours and 20 hours respectively.
FIG. 7 is a graph showing the results of data on RFU values of fluorescent proteins synthesized by the lactose + glucose + maltodextrin protein synthesis system in example 5 of the present invention; wherein the lactose concentration is 20-200 mM, the maltodextrin concentration is 320 mM, the glucose concentration is 20mM, and the detection time is 3 hours and 20 hours respectively.
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, lactose and 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 system 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. Compared with a reaction system taking glucose as an energy source, the reaction system taking maltodextrin as an energy source has the RFU value increased by about 1 time. Compared with a reaction system taking glucose as an energy source, the RFU value of the reaction system taking lactose as the energy source is increased by more than 3 times. Compared with a reaction system taking glucose as an energy source, the RFU value of the reaction system taking lactose and glucose as the energy source is increased by about 1 time. Compared with a reaction system taking glucose as an energy source, the reaction system taking lactose, glucose and maltodextrin has the RFU value increased by about 4 times. Based on the method, the invention provides a high-efficiency and low-cost protein synthesis system.
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; (b) a saccharide which is maltodextrin, lactose, or a combination of maltodextrin and glucose, or a combination of lactose and glucose, or a combination of maltodextrin and lactose and glucose; (c) a phosphoric acid compound.
Preferably, the protein synthesis system further comprises an active enzyme capable of catalyzing the metabolism of carbohydrates to produce ATP. 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 protein synthesis system further comprises one or more components of the group consisting of: (d1) polyethylene glycol; (d2) a substrate for RNA synthesis; (d3) a substrate for synthesizing a protein; (d4) magnesium ions; (d5) potassium ions; (d6) a buffering agent; (d7) an RNA polymerase; (d8) dithiothreitol (DTT); (d9) optionally a solvent, which is 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 substrate of the synthetic protein 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.
Further, the buffer is selected from the group consisting of: 4-hydroxyethyl piperazine ethanesulfonic acid, tris (hydroxymethyl) aminomethane or a combination thereof.
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. Where the concentrations of the nucleoside triphosphate mixture and the amino acid mixture are described in the various examples, the concentrations refer to the concentrations of the individual species in the mixture, not the total species 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), 0.1 mM 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, and finally 50% by volume of yeast cell extract.
In vitro protein synthesis reaction: adding 15 ng/muL enhanced green fluorescent protein DNA into the reaction system, uniformly mixing, and placing in an environment 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.
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 the enhanced green fluorescent protein is 1.50 mu g/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), 0.1 mM 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, and finally 50% by volume of yeast cell extract.
In vitro protein synthesis reaction: adding 15 ng/muL enhanced green fluorescent protein DNA into the reaction system, uniformly mixing, and placing in an environment of 20-30 ℃ for reaction.
Fluorescent protein activity assay: 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.
FIG. 1 is a graph showing the results of data on RFU values of fluorescent proteins synthesized in the maltodextrin + glucoprotein synthesis system of the present 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. 1: 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. The yield of the enhanced green fluorescent protein is 121.10 mu g/mL.
The addition of alpha amylase is beneficial to hydrolysis of maltodextrin, and in this example, in the absence of alpha amylase, 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 μ g/mL.
FIG. 2 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 influence of glucose at different 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. 2: the highest RFU value of the fluorescent protein is achieved when the glucose concentration is about 20 mM.
Comparative example 2 in vitro cell-free protein Synthesis System containing glucose
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, 0.1 mM amino acid mixture, 1.7 mM dithiothreitol, 0-80 mM glucose, 20mM tripotassium phosphate, 0.03 mg/mL T7 RNA polymerase, 2% polyethylene glycol, and finally 50% by volume of yeast cell extract.
In vitro protein synthesis reaction: adding 15 ng/muL enhanced green fluorescent protein DNA into the system, mixing uniformly, and placing in an environment 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. 3 is a graph showing the result of data on RFU values of fluorescent proteins synthesized by the individual glucose protein synthesis system in this example; wherein the glucose concentration is 0-80 mM, and the detection time is 3 hours. As can be seen from FIG. 3, the addition of glucose at various concentrations significantly increased the yield of the target protein, which was RFU 280 at a glucose concentration of 20mM for 3 hours. The yield of the enhanced green fluorescent protein is 16.02 mu g/mL. After 3 hours of reaction, the RFU value began to decline slowly.
EXAMPLE 2 in vitro cell-free protein Synthesis System with maltodextrin
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, 0.1 mM amino acid mixture, 1.7 mM dithiothreitol, 0-500 mM maltodextrin, 20mM tripotassium phosphate, 0.002mg/mL alpha amylase, 0.03 mg/mL T7 RNA polymerase, 2% polyethylene glycol, and finally 50% by volume of yeast cell extract.
In vitro protein synthesis reaction: adding 15 ng/muL enhanced green fluorescent protein DNA into the system, mixing uniformly, and placing in an environment 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. 4 is a graph showing the results of data on RFU values of fluorescent proteins synthesized by the single maltodextrin protein synthesis system in this example; wherein the concentration of maltodextrin is 0-500 mM, and the detection time is 20 hours. As can be seen from FIG. 4, the addition of maltodextrin at various concentrations significantly increased the yield of the target protein, which was a 20 hour reaction at a maltodextrin concentration of 164mM, and the RFU value of 620. Enhanced green fluorescent protein production 37.04. mu.g/mL.
Example 3 in vitro cell-free protein Synthesis System containing lactose
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, 0.1 mM amino acid mixture, 1.7 mM dithiothreitol, 0-200 mM lactose, 20mM tripotassium phosphate, 0.03 mg/mL T7 RNA polymerase, 2% polyethylene glycol, and finally 50% by volume of yeast cell extract.
In vitro protein synthesis reaction: adding 15 ng/muL enhanced green fluorescent protein DNA into the system, mixing uniformly, and placing in an environment 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. 5 is a graph showing the results of data on RFU values of fluorescent proteins synthesized by the single lactoalbumin synthesis system in this example; wherein the lactose concentration is 0-200 mM, and the detection time is 3 hours and 20 hours respectively. As can be seen from FIG. 5, the yield of the target protein was significantly improved by adding lactose at various concentrations, with the most significant effect at a lactose concentration of 200 mM (20 hr RFU from 25 to 1300). Enhanced green fluorescent protein production was 81.26. mu.g/mL.
Example 4 in vitro cell-free protein Synthesis System containing lactose + glucose
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, 0.1 mM amino acid mixture, 1.7 mM dithiothreitol, 15 mM glucose, 0-400 mM lactose, 20mM tripotassium phosphate, 0.03 mg/mL T7 RNA polymerase, 2% polyethylene glycol, and finally 50% by volume of yeast cell extract.
In vitro protein synthesis reaction: adding 15 ng/muL enhanced green fluorescent protein DNA into the system, uniformly mixing, and placing in an environment at 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. 6 is a graph showing the results of data on RFU values of fluorescent proteins synthesized by the lactose + glucose protein synthesis system in this example; wherein the lactose concentration is 0-400 mM, the glucose concentration is 15 mM, and the detection time is 3 hours and 20 hours respectively. As can be seen from FIG. 6, the yield of the target protein was significantly improved by adding lactose at various concentrations, with the most significant effect at a lactose concentration of 178 mM and an RFU value of 600 at 20 hours of reaction. The yield of the enhanced green fluorescent protein is 35.78 mu g/mL.
Example 5 in vitro cell-free protein Synthesis System containing lactose + glucose + maltodextrin
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, 0.1 mM amino acid mixture, 1.7 mM dithiothreitol, 20mM glucose, 0-200 mM lactose, 320 mM maltodextrin, 20mM tripotassium phosphate, 0.03 mg/mL T7 RNA polymerase, 0.002mg/mL alpha amylase, 2% polyethylene glycol, and finally 50% by volume of yeast cell extract.
In vitro protein synthesis reaction: adding 15 ng/muL enhanced green fluorescent protein DNA into the system, mixing uniformly, and placing in an environment 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. 7 is a graph showing the results of data on RFU values of fluorescent proteins synthesized in the lactose + glucose + maltodextrin protein synthesis system of this example; wherein the lactose concentration is 20-200 mM, the maltodextrin concentration is 320 mM, the glucose concentration is 20mM, and the detection time is 3 hours and 20 hours respectively. As can be seen from FIG. 7, the yield of the target protein was significantly improved by adding lactose at various concentrations, with the most significant effect at a lactose concentration of 40 mM and an RFU value of 1550 at 20 hours of reaction. The yield of the enhanced green fluorescent protein is 97.76 mu g/mL.
Results of the experiment
As can be seen from examples 1,2, 1 and 2, the cell-free protein synthesis system using glucose and maltodextrin as energy sources (example 1) has a protein synthesis capacity improved by more than 30 times compared with the synthesis system using phosphocreatine + phosphocreatine kinase (comparative example 1) as energy sources; compared with a synthesis system (comparative example 2) taking glucose as an energy source, the protein synthesis capacity of the protein synthesis system is improved by more than 5 times; compared with the synthesis system using maltodextrin (example 2) as energy source, the protein synthesis capacity of the protein synthesis system is improved by more than 2 times.
Compared with a synthesis system taking phosphocreatine and phosphocreatine kinase (comparative example 1) as an energy source, the cell-free protein synthesis system taking maltodextrin (example 2) as the energy source has the protein synthesis capacity improved by about 10 times; compared with a synthetic system (comparative example 2) taking glucose as an energy source, the protein synthesis capacity of the protein is improved by about 1 time.
As can be seen from example 3, comparative example 1, and comparative example 2, the cell-free protein synthesis system using lactose (example 3) as an energy source has a protein synthesis capacity improved by more than 20 times compared with the synthesis system using phosphocreatine + phosphocreatine kinase (comparative example 1) as an energy source; compared with a synthetic system (comparative example 2) taking glucose as an energy source, the protein synthesis capacity of the protein is improved by more than 3 times.
As can be seen from example 4, comparative example 1 and comparative example 2, the cell-free protein synthesis system using lactose + glucose (example 4) as an energy source has about 10 times higher protein synthesis capacity than the synthesis system using phosphocreatine + phosphocreatine kinase (comparative example 1) as an energy source; compared with a synthetic system (comparative example 2) taking glucose as an energy source, the protein synthesis capacity of the protein is improved by about 1 time.
As can be seen from example 5, comparative example 1 and comparative example 2, the cell-free protein synthesis system using lactose + glucose + maltodextrin (example 5) as an energy source has about 25 times higher protein synthesis capacity than the synthesis system using phosphocreatine + phosphocreatine kinase (comparative example 1) as an energy source; compared with a synthetic system (comparative example 2) taking glucose as an energy source, the protein synthesis capacity of the protein is improved by about 4 times.
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 any changes and modifications made are within the scope of the invention.
Claims (11)
1. An in vitro cell-free protein synthesis system, comprising:
(a) a kluyveromyces cell extract;
(b) a saccharide selected from lactose, or a combination of maltodextrin and glucose, or a combination of maltodextrin and lactose and glucose;
(c) a phosphoric acid compound selected from orthophosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, or combinations thereof;
the in vitro cell-free protein synthesis system further comprises an active enzyme capable of catalyzing the metabolism of carbohydrates to produce ATP; the active enzyme is amylase, galactosidase, phosphorylase, glucose phosphate mutase, or a combination thereof;
when the saccharide substance comprises glucose, the concentration of the glucose is 8.8-128 mmol/L;
when the saccharide substance comprises maltodextrin, the concentration of the maltodextrin is 84-500 mmol/L.
2. The in vitro cell-free protein synthesis system of claim 1, wherein the active enzyme is galactosidase, phosphorylase, phosphoglucomutase, or a combination thereof; the saccharide is lactose or the combination of maltodextrin, lactose and glucose.
3. The in vitro cell-free protein synthesis system of claim 1, wherein the amylase is an alpha amylase; the saccharide is the combination of maltodextrin and glucose or the combination of maltodextrin, lactose and glucose.
4. The in vitro cell-free protein synthesis system of claim 1, further comprising one or more components of the group consisting of:
(d1) polyethylene glycol;
(d2) a substrate for RNA synthesis;
(d3) a substrate for synthesizing a protein;
(d4) magnesium ions;
(d5) potassium ions;
(d6) a buffering agent;
(d7) an RNA polymerase;
(d8) dithiothreitol;
(d9) optionally a solvent, which is water or an aqueous solvent.
5. The in vitro cell-free protein synthesis system of claim 1, wherein the kluyveromyces is kluyveromyces lactis.
6. The in vitro cell-free protein synthesis system of claim 1, wherein the concentration v/v of the cellular extract is from 20% to 70% based on the total volume of the in vitro cell-free protein synthesis system.
7. The in vitro cell-free protein synthesis system of claim 6, wherein the concentration v/v of the cellular extract is from 30% to 60% based on the total volume of the in vitro cell-free protein synthesis system.
8. The in vitro cell-free protein synthesis system of claim 7, wherein the concentration v/v of the cell extract is from 40% to 50% based on the total volume of the in vitro cell-free protein synthesis system.
9. A kit comprising a container and the components of the in vitro cell-free protein synthesis system of any one of claims 1-8 disposed in the container.
10. 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 8;
(ii) adding a DNA molecular template encoding the foreign protein, and carrying out incubation reaction under a suitable condition, thereby synthesizing the foreign protein;
(iii) optionally, isolating or detecting the foreign protein from the in vitro cell-free protein synthesis system.
11. The method for synthesizing an exogenous protein according to claim 10, wherein the incubation reaction is carried out under a condition of 20 to 30 ℃.
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