阅读说明：本技术 含咪唑的体外无细胞蛋白合成体系及其应用 (Imidazole-containing in-vitro cell-free protein synthesis system and application thereof ) 是由 郭敏 章小铃 于雪 于 2019-03-27 设计创作，主要内容包括：本发明提供一种含有咪唑的体外无细胞蛋白合成体系,其能够提高体外无细胞蛋白合成的能力。其无需从分子水平进行改造,体系组成成分来源常规,能够达到节约成本、操作简单、应用范围广泛的效果。相应的,提供还有咪唑的无细胞蛋白合成体系的应用,具体的为咪唑在提高蛋白表达量的无细胞蛋白合成体系中的应用及利用该体系合成外源蛋白的方法。(The invention provides an imidazole-containing in vitro cell-free protein synthesis system, which can improve the capacity of in vitro cell-free protein synthesis. The method does not need to be modified from the molecular level, and the system components are conventional in source, so that the effects of cost saving, simple operation and wide application range can be achieved. Correspondingly, the application of the cell-free protein synthesis system of imidazole is also provided, in particular to the application of imidazole in the cell-free protein synthesis system for improving the protein expression level and a method for synthesizing foreign protein by using the system.)

1. A cell-free protein synthesis system for increasing the expression level of a protein, which is characterized in that: adding imidazole to a cell-free protein synthesis system, wherein the imidazole does not function as an antibiotic in the synthesis system.

2. The synthesis system according to claim 1, characterized in that: the imidazole is present at a concentration of at least 0.199 mM.

3. The synthesis system according to claim 1, characterized in that: the cell-free protein synthesis system comprises a cell extract, RNA polymerase, a nucleotide mixture and an amino acid mixture.

4. The synthesis system according to claim 3, characterized in that: the system further comprises one or more components of a buffer, potassium ions, magnesium ions, dithiothreitol, polyethylene glycol, optionally an aqueous solvent, and an energy regeneration system.

5. The synthesis system according to any one of claims 3 or 4, characterized in that: the cell source of the cell extract is selected from one of prokaryotic cells and eukaryotic cells or the combination of the prokaryotic cells and the eukaryotic cells.

6. The synthesis system according to claim 5, characterized in that: the cell source of the cell extract is selected from one of Escherichia coli, mammalian cells, plant cells, yeast cells, insect cells or their combination.

7. The synthesis system according to claim 6, characterized in that: the yeast cell is selected from the group consisting of: one or the combination of saccharomyces cerevisiae, pichia pastoris and kluyveromyces.

8. The synthesis system according to claim 7, characterized in that: the Kluyveromyces is selected from one of Kluyveromyces lactis, Kluyveromyces marxianus and Kluyveromyces polybracteus or the combination thereof.

9. An application of imidazole in cell-free protein synthesis system for increasing protein expression level.

10. A method for synthesizing a foreign protein by using a cell-free protein synthesis system is characterized by comprising the following steps: the method comprises the following steps:

(i) providing a cell-free protein synthesis system according to any one of claims 1 to 8;

(ii) adding a DNA template for encoding the foreign protein, and reacting to synthesize the foreign protein;

further comprising the optional step (iii) of isolating or detecting said foreign protein.

Technical Field

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

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).

Imidazoles (imidazoles) are common antifungal agents. The action mechanism is to inhibit the biosynthesis of ergosterol of fungal cell membranes. It can selectively inhibit 14-alpha-demethylase dependent on fungal cytochrome P-450, accumulate 14-alpha-methyl sterol, make ergosterol on cell membrane unable to synthesize, and make cell membrane permeability change, so as to make the fungus die. Imidazole reported in the prior literature or patent is used as a purification reagent or an elution reagent component in the protein production process, and mainly plays a role in purifying and eluting proteins (such as CN101479379B and CN 105283556A).

With the development of science and technology, a cell-free expression system is also called an in vitro protein synthesis system, exogenous target mRNA or DNA is taken as a protein synthesis template, and substances such as a substrate required by protein synthesis and transcription and translation related protein factors are supplemented through manual control, so that the synthesis of target protein can be realized (3), and the cell-free expression system is a rapid, time-saving and convenient protein expression mode. Although cell-free protein synthesis systems have many advantages, the supply of energy and reaction time to synthesize proteins in large quantities is a critical issue affecting the efficiency and cost of the system (4). However, problems that may be encountered in extending the reaction time for in vitro protein synthesis are the synthesis of nonspecific substances, and the consumption of substrates, energy, and the like required for protein synthesis. Therefore, increasing the level of protein synthesis by cell-free protein synthesis via other pathways is a problem that needs to be addressed.

In view of the above, the present invention provides a novel in vitro cell-free protein synthesis system, which can improve the capacity of cell-free protein synthesis in vitro.

Reference to the literature

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

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

3. Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinantprotein expression for use in pharmaceutical research. Curr Opin Struct Biol[Internet]. 2013;23(3):393–402.

4. Anne Zemella,Lena Thoring, Christian Hoffmeister,and StefanKubick.Cell-Free Protein Synthesis:Pros and Cons of Prokaryotic and EukaryoticSystems. Chembiochem. 2015, 16:2420-2431.

Disclosure of Invention

The invention provides a brand-new in vitro cell-free protein synthesis system which can improve the capacity of in vitro cell-free protein synthesis. The method does not need to be modified from the molecular level, and the system components are conventional in source, so that the effects of cost saving, simple operation and wide application range can be achieved.

The invention includes the following aspects:

in a first aspect, there is provided a cell-free protein synthesis system for increasing the expression level of a protein, wherein imidazole is added to the cell-free protein synthesis system, and wherein the imidazole does not function as an antibiotic in the synthesis system.

Further, the imidazole is at a concentration of at least 0.199 mM.

Further, the concentration of imidazole is 0.199-2.27 mM.

Further, the concentration of imidazole is 0.448 mM.

Further, the cell-free protein synthesis system comprises a cell extract, RNA polymerase, a nucleotide mixture and an amino acid mixture.

Further, the system may further comprise one or more of a buffer, potassium ions, magnesium ions, dithiothreitol, polyethylene glycol, and an energy regeneration system.

Further, the cell source of the cell extract is selected from one of prokaryotic cells, eukaryotic cells or a combination thereof.

In another preferred embodiment, the cell extract is derived from one or more types of cells selected from the group consisting of: escherichia coli, mammalian cells (such as HF9, Hela, CHO, HEK293), plant cells, yeast cells, insect cells.

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

In another preferred embodiment, the yeast cell is selected from the group consisting of: saccharomyces cerevisiae, saccharomyces pichia, kluyveromyces, or combinations thereof; preferably, the kluyveromyces cell comprises: kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces polybracteus, preferably Kluyveromyces lactis.

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 RNA polymerase is not particularly limited, and may be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.

Wherein the magnesium ion is derived from a magnesium ion source, which is selected from the group consisting of: one or the combination of magnesium acetate, magnesium glutamate and magnesium citrate.

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

Wherein the energy regeneration system is selected from the group consisting of: one of a phosphocreatine/phosphocreatine system, glycolysis pathway and its intermediates energy system, polysaccharide and phosphate energy system, saccharides or combinations thereof.

Wherein the buffer is selected from the group consisting of: one or a combination of Tris-HCl, Tris base, HEPES, Tris-citric acid, citric acid-citrate, Tris-citrate.

Wherein, the concentration of polyethylene glycol or its analogue is not particularly limited, and usually, the concentration (w/v) of polyethylene glycol or its analogue is 0.1 to 8%, preferably 0.5 to 4%, more preferably 1 to 2%, based on the total weight of the protein synthesis system. Representative PEGs are selected from the group consisting of: one or a combination of PEG3000, PEG8000, PEG6000 and PEG 3350.

Wherein the polyethylene glycol comprises polyethylene glycol with molecular weight (Da) of 200-10000, such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc., preferably, polyethylene glycol with molecular weight of 3000-10000.

Wherein, the nucleotide mixture is selected from one of nucleoside monophosphate, nucleoside triphosphate and deoxynucleotide or the combination thereof.

The amino acid mixture includes at least 20 kinds of amino acids, such as glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine. The amino acid may also include an unnatural amino acid, may also include a radioisotope-labeled amino acid, and further, may also include a modified amino acid.

Wherein, the imidazole does not play an antibiotic function in a synthesis system and is identified by a fungus culture medium. The fungus culture medium is selected from Sabouraud's dextrose glycoprotein peptone agar medium without antibiotic, potato dextrose agar medium, polysorbate 80 medium, corn Tween 80 agar medium, malt extract agar medium, yeast extract peptone dextrose agar medium (YPD or YEPD medium).

In a second aspect, an imidazole is used in a cell-free protein synthesis system for increasing the expression level of a protein.

In a third aspect, there is provided a method for synthesizing a foreign protein using a cell-free protein synthesis system, comprising: the method comprises the following steps:

(i) providing a cell-free protein synthesis system according to any one of the first aspect;

(ii) adding a DNA template for encoding the foreign protein, and reacting to synthesize the foreign protein;

further comprising the optional step (iii) of isolating or detecting said foreign protein.

Further, suitable conditions for the reaction include reaction temperatures in the range of 10 ℃ to 40 ℃, preferably 20 ℃ to 35 ℃, preferably 20 ℃ to 30 ℃, more preferably 25 ℃.

Further, the reaction time is 1 to 72 hours, preferably 2 to 24 hours, more preferably 3 to 12 hours; the reaction time can be determined artificially according to specific conditions (such as the amount of a reaction substrate, the content of expected obtained protein and the like), and can also be 3-15h, 3-20h, and specific time points, such as 3h, 5h, 10h, 15h, 18h and 20 h.

The invention has the advantages and beneficial effects that:

(1) on the basis of no need of molecular modification, imidazole is added into a cell-free protein synthesis system to improve the protein synthesis capacity of the in vitro cell-free protein synthesis system;

(2) the imidazole is not proved to play an antifungal role in a cell-free protein synthesis system, and the safety of the system is also demonstrated while the imidazole is excluded to play an antibacterial role;

(3) the imidazole-containing cell-free protein synthesis system is simple to operate, saves cost, has universality and can be used for any cell-free protein synthesis system.

Drawings

FIG. 1 is a graph showing the effect of imidazole concentration on an in vitro protein synthesis system, where PC is a positive control without imidazole and NC is a negative control without imidazole and DNA template.

FIG. 2 is a diagram showing the results of fungal culture identification.

Detailed Description

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. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight. The invention takes Kluyveromyces lactis (K. lactis or kl) as the cell extract source in the embodiment, but the same design, analysis and experimental methods are also suitable for other yeast cells, animal cells and other eukaryotic cells (mammalian cells, plant cells and insect cells) and prokaryotic cells (such as escherichia coli).

Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

Technical terms

In the present invention, the expression of the cell extract, cell lysate, and cell lysate is the same.

Comparative example 1 imidazole-free in vitro cell-free protein Synthesis System

In vitro cell-free protein synthesis reaction system: trimethylolaminomethane (Tris-HCl) at a final concentration of 9.78 mM, pH 8.0, 80 mM potassium acetate, 5.0 mM magnesium acetate, 1.5 mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uridine triphosphate, each at a concentration of 1.5 mM), 0.7mM 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 0.7mM), 1.7mM Dithiothreitol (DTT), 2% polyethylene glycol, 15mM glucose, 24mM tripotassium phosphate, 50% by volume of yeast cell extract, 15 ng/μ L enhanced green fluorescent protein (eGFP) 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 for 20 hours.

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.