RU2802080C1 - Cell-free protein synthesis system based on staphylococcus aureus cells, a method of protein synthesis based on staphylococcus aureus cells using a cell-free protein synthesis system based on staphylococcus aureus cells and a method of detecting protein synthesis inhibitors using it - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: proposed cell-free protein synthesis system based on Staphylococcus aureus cells, obtained by mixing the components of the reaction mixture in the specified order and volume ratio (vol.%); a method of protein synthesis in a cell-free system based on Staphylococcus aureus cells, including incubation of the claimed cell-free system at 37°C; a method of detecting inhibitors of protein synthesis based on Staphylococcus aureus cells, including incubation at 37°C potential inhibitors of protein synthesis in conjunction with a cell-free protein synthesis system based on Staphylococcus aureus cells, quantitative measurement of the synthesized protein by a luminescent or fluorescent radiation signal and determination of the ability to inhibit protein synthesis by the incidence of a light signal.

EFFECT: inventions expand the arsenal of agents for protein synthesis in open systems outside cells based on cellular metabolism, for screening antimicrobial substances in biotechnological and pharmaceutical industries.

6 cl, 2 dwg, 3 ex

Description

The claimed group of technical solutions relates to the field of molecular biology and synthetic biology, specifically to a means and method for protein synthesis in open systems outside cells based on cellular metabolism, screening of antimicrobial substances, including those active against the Staphylococcus aureus translation apparatus. The claimed technical solutions will make it possible to search for chemical compounds for the ability to inhibit protein synthesis, including Staphylococcus aureus bacteria, to perform functional tests of bacterial proteins, including Staphylococcus aureus bacteria proteins, to perform the synthesis of bacterial proteins for biotechnology and structural biology. The inventions can be used, for example, in biotechnological and pharmaceutical industries, in research organizations, in order to carry out reactions of protein synthesis of Staphylococcus aureus bacteria in a natural environment in vitro, to test the effect of chemicals (for example, antibiotics) on Staphylococcus aureus protein biosynthesis. The synthesized protein using the claimed cell-free system can be used in biotechnology, including after its isolation, purification and modification of the target protein.

Further in the text, the applicant gives the terms that are necessary to facilitate an unambiguous understanding of the essence of the claimed materials and to exclude contradictions and / or controversial interpretations when performing an examination on the merits.

ATP is adenosine triphosphate.

HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

EDTA - Ethylenediaminetetraacetic acid.

NTP , nucleotide triphosphate.

CTP - cytidine triphosphate.

GTP is guanosine triphosphate.

UTP - uridine triphosphate.

RNasine is an RNase inhibitor.

To solve the problem of antibiotic resistance of strains of pathogenic microorganisms, it is necessary to develop new drugs based on an integrated approach. One of the most traditional ways of searching for antimicrobial drugs is sequential testing of new natural or synthetic compounds on culture liquids, after which the chemical and spatial structure is determined and the mechanism of action of the inhibitor is searched. At the same time, the search for a molecular target in a pathogen cell, which is affected by the test substance with antimicrobial activity, is one of the most difficult stages.

At the present stage of development of pharmacology, and biotechnology based on it, one of the key tasks is to obtain data on the relationships and interactions of the parts that make up a biological system, as well as its kinetic parameters - for this, in vitro approaches are often used and, in particular, cell-free systems.

Programming cell-free systems provides many benefits:

- ease of experimentation,

- the ability to control the concentrations and activities of genes and enzymes,

- ease of obtaining quantitative measurements,

- the ability to control a large number of parameters that can be estimated using high-throughput (high-throughput) approaches.

In a general sense, a cell-free protein synthesis system is a cell without a membrane, placed in a quasi-natural environment, consisting of a buffer system, metabolites and enzymes that help metabolism. In a cell-free protein synthesis system, a set of chemical reactions are reproduced that reflect the main metabolic processes necessary for protein synthesis. The cell-free protein synthesis system includes all the components necessary for transcription, translation, as well as ATP synthesis systems that provide energy for biochemical reactions.

The cell-free protein synthesis system consists of four main components:

-cell extract as the basis of metabolism,

-DNA/RNA,

-substrates for transcription and translation,

- ATP regeneration systems.

The components joined together create the conditions for the flow of the main stages of protein synthesis in vitro . Due to the presence of genetic features in the structure of the protein synthesis apparatus in each type of organism, it is necessary to use the appropriate cell extract for a particular organism and select individual conditions and concentrations of the components of cell-free protein synthesis systems.

To screen for potential inhibitors, the test substance is added to the reaction mixture of the cell-free protein synthesis system and the level of inhibition of protein synthesis is assessed by the amount of signal protein mediated through the fluorescence or luminescence signal. At the same time, due to the openness of the system, it is possible to carry out large-scale verification and optimization by easily changing the experimental conditions, for example, the concentration of the added inhibitor substance, within the framework of one experiment. Also, within the framework of a cell-free protein synthesis system, it is possible to separate transcription from translation and evaluate the effect on individual processes, thereby clarifying the scope of the inhibitor substance. Cell-free protein synthesis systems have the advantages of a reaction rate (from 2 to 10 hours), the ability to automatically carry out a large number of measurements at a time, the ability to control the reaction due to the absence of a cell wall, the ability to add any proteins, including those toxic to the cell.

Currently, cell-free protein synthesis systems are actively used in the field of synthetic biology to create model minimal programmable systems, design proteins, search for biocatalysts, for large-scale production of target proteins and molecules, and to study the processes of protein synthesis inhibition.

The high cost of cell-free systems has previously made them unattractive for high throughput screening. Recently, however, cell-free systems have attracted the attention of researchers from other fields, especially from the field of synthetic biology. Now the technology is used in the field of synthetic biology to create model minimal programmable systems, design proteins, search for biocatalysts, for large-scale development of target proteins and molecules, and search for inhibitors. Unlike test systems based on cell cultures, in a cell-free protein synthesis system, it is possible to conduct accurate kinetic experiments with the addition of substances.

Thus, the sources of "An Escherichia coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells" are known (Cell-free expression system based on E. coli cells and its use in synthetic gene chains and artificial cells) [Shin J. Et al. An Escherichia coli Cell-Free Expression Toolbox: Application to Synthetic Gene Circuits and Artificial Cells // ACS Synth. Biol, 2012. Vol.1. N. 1. P. 29–41], “A cell-free protein synthesis system for high-throughput proteomics” [Sawasaki T. et al. A cell-free protein synthesis system for high-throughput proteomics // Proc Natl Acad Sci USA, 2002. Vol. 99(23). P. 14652-14657], "Microscale Adaptation of In Vitro Transcription/Translation for High-Throughput Screening of Natural Product Extract Libraries" [Lowell, AN et al. Microscale Adaptation of In Vitro Transcription/Translation for High-Throughput Screening of Natural Product Extract Libraries // Chem. Biol. Drug Des. 2015. Vol. 86. P. 1331–1338. The essence is cell-free systems for testing regulatory elements and protein synthesis in the gram-negative bacterium Escherichia coli .

The disadvantage of these systems is the limited use only for gram-negative bacteria Escherichia coli and the impossibility of synthesizing modified proteins of the bacterium Staphylococcus aureus due to the absence of transcription and translation components specific for Staphylococcus aureus .

Known source "Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements" (Development of a cell-free transcription-translation system based on Bacillus subtilis cells for prototyping regulatory elements) [Kelwick R. et al. Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements // Metab. Eng, 2016. Vol. 38. P. 370-381]. The entity is a cell-free protein synthesis system based on B. subtilis strain WB800N capable of producing 0.8 μM of the fluorescent GFP protein. The possibility of creating a library of B. subtilis promoters based on the wild-type Pgrac (σA) promoter, which demonstrate a number of comparable transcriptional activities in vitro and in vivo , has been shown.

The disadvantage of this system is the limited use only for the bacterium Bacillus subtilis , which does not allow the synthesis of modified proteins of the bacterium Staphylococcus aureus , due to the absence of transcription and translation components specific for Staphylococcus aureus .

So, the invention is known according to the patent of the Russian Federation No. 2148649 "Method for obtaining polypeptides in a cell-free system (variants) and a device for its implementation." The essence is a method for obtaining polypeptides in a cell-free system, which involves preparing a reaction mixture and a feed mixture, installing two porous barriers inside the reaction module that define the boundaries of the reaction volume, introducing the reaction mixture into the reaction volume and carrying out synthesis under specified conditions, maintaining synthesis by introducing a feed mixture into during the synthesis process, synthesized target polypeptides are removed from the reaction volume or left inside the reactor, characterized in that before the synthesis, a mixture of consumable high-molecular components is prepared, the ratio of the volume of the feed mixture and consumable high-molecular components to the volume of the fraction containing the target polypeptide is selected, the feed mixture input mode is set, and of consumable high molecular weight components into the reaction volume, during the synthesis, the synthesis products are separated into a fraction containing low molecular weight components and a fraction containing high molecular weight components of the synthesis, including the target polypeptide, and the main part of the fraction containing low molecular weight components is removed from the reaction volume through one or both parts second porous barrier.

The disadvantage of the known technical solution is the time limit for the biosynthesis of proteins due to the leaching of the components of the cell-free system that are absent in the feed mixture.

An invention is known according to the RF patent No. 2162104 "Method of synthesizing a fluorescently labeled protein". The essence is a method for synthesizing a fluorescently labeled protein in a prokaryotic cell-free protein synthesis system, which includes the following steps: (a) incubation of a sample of ribosomes obtained from a cell-free extract with plasmid DNA containing a coding sequence for a protein of interest, and the sample is incubated in a medium for conjugated transcription/translation together with a fluorescently labeled aminoacyl-tRNA, (b) partial purification of said fluorescently labeled protein by separating the newly synthesized fluorescently labeled protein from other fluorescent components in this sample, (c) measuring the amount of synthesized protein, (d) determining fluorescence of the newly synthesized protein; (e) determination of the biological activity of the newly synthesized protein.

The disadvantage of the known technical solution is the need for an additional step of purification of the synthesized fluorescently labeled protein from other fluorescent components in the sample.

An invention is known according to RF patent No. 2620074 "Recombinant plasmid DNA pDUALREP2 and a strain transformed by it to identify substances and mixtures that inhibit protein biosynthesis and/or cause an SOS response in bacteria." The essence is recombinant plasmid DNA pDualrep2, designed to identify compounds that inhibit protein biosynthesis and/or cause an SOS response in bacteria, including: an origin of replication capable of maintaining a stable number of copies of the plasmid in the cell; a selective marker for selecting cells containing the plasmid; genes for fluorescent proteins with distinct RFP and Katushka2S fluorescence spectra; an attenuator of the tryptophan operon, in which the UGG tryptophan codons were replaced with the GCG alanine codons; E. coli sulA gene promoter activated upon inactivation of the SOS response repressor LexA.

The disadvantage of the known technical solution is the limited use only for gram-negative Escherichia coli bacteria and the impossibility of synthesizing modified proteins of the bacterium Staphylococcus aureus due to the absence of transcription and translation components specific to Staphylococcus aureus .

The invention is known (WO 2019035955 A1 publ. 21.02.2019) "High-throughput system using a cell-free expression system and in situ sequencing". The essence is a method for screening a plurality of template nucleic acid sequences for the production of small molecules, including adding a template nucleic acid sequence from a plurality to each reaction volume from a plurality of reaction volumes, each reaction volume includes a cell-free expression system under conditions of transcription and translation; detecting the formation of a small molecule in the target reaction volume; and sequencing the template nucleic acid sequence of the target reaction volume.

The disadvantage of the known technical solution is the use of cell lysates from the actinobacteria Streptomyces lividans , Streptomyces violaceoruber, Streptomyces coelicolor, Streptomyces albovinaceus, gram-negative bacteria Vibrio natriegens , Escherichia coli, Pseudomonas aeruginosa and the lack of a description of the optimized protein synthesis protocol in the gram-positive bacterium Staphylococcus aure us .

A known source is " Staphylococcus aureus cell extract transcription-translation assay: firefly luciferase reporter system for evaluating protein translation inhibitors" [Murray RW et al. Staphylococcus aureus cell extract transcription-translation assay: firefly luciferase reporter system for evaluating protein translation inhibitors // Antimicrob Agents Chemother. 2001 Vol. 45(6). P. 1900-1904], which describes the Staphylococcus aureus reporter system, consisting of a lysate of an extract of Staphylococcus aureus strain RN4220 cells grown on a nutrient medium Brain heart, previously sequentially washed in 500 ml of cold buffer A (10 mM Tris-acetate, pH 8.0 , 14 mM Mg-acetate, 1 mM dithiothreitol) containing 1 M KCl, followed by 250 ml of buffer A containing 50 mM KCl, after which the cell suspension was resuspended to a final volume of 99 ml of buffer B (10 mM Tris-acetate, pH 8 ,0, 20 mM Mg-acetate, 50 mM KCl, 1 mM dithiothreitol) followed by incubation of the solution with a commercial preparation of the enzyme lysostaphin to destroy the cell wall, then the suspension was incubated at 37 °C for 45-60 minutes, after which the solution was precipitated three times centrifugation at 4°C at 16,000 rpm (30,000 3 g) for 30 min and two-thirds of the supernatant was removed, after which 0.25 volumes of preincubation buffer (670 mM Tris-acetate, pH 8.0, 20 mM Mg-acetate, 7 mM Na3-phosphoenolpyruvate, 7 mM dithiothreitol, 5.5 mM ATP, 70 mM mixture of 20 proteinogenic amino acids, 75 mg/ml pyruvate kinase) and the mixture was incubated at 37°C for 30 min to increase active ribosomes after they read all available mRNA in solution; after which, the additionally incubated supernatant was dialyzed for 12 hours at 4°C in 2 L of dialysis buffer (10 mM Tris-acetate, pH 8.0, 14 mM Mg-acetate, 60 mM K-acetate, 1 mM dithiothreitol) with a single change buffer; for concentration to 10 mg/mL, the (purified) lysate was dialyzed in bags (Spectra-Por 7; LMW 3500 Da) containing pre-chilled polyethylene glycol 8000 (Sigma) at 4°C; the resulting reaction mixture was supplemented with a PCR product containing the luciferase gene under the control of the Staphylococcus aureus (cap1A) capsular polysaccharide synthesis gene promoter and the Luciferase Assay Reagent (Promega Corporation) commercial kit for the luminescent protein synthesis reaction; at the final stage, a luminescence signal was recorded.

The disadvantages of the known system are the need for a stage of incubation of the lysate to increase active ribosomes after reading all the available mRNA in solution, the need to use a commercial preparation of lysostaphin for cell lysis, the need to add a commercial set of Luciferase Assay Reagent (Promega Corporation) to the reaction mixture to be able to register the luminescence signal , the lack of the possibility of synthesis of a fluorescent protein and the possibility of registering fluorescence.

The technical problem to be solved by the claimed group of inventions is the expansion of the arsenal of means and methods for protein synthesis, as well as for screening substances for the ability to inhibit protein synthesis of bacteria, including Staphylococcus aureus .

The technical result of the claimed group of inventions is

– the possibility of synthesizing proteins of the gram-positive bacterium Staphylococcus aureus in a natural genetic and metabolic environment, which makes it possible to obtain functional proteins of Staphylococcus aureus with post-translational modifications due to the presence of all the necessary Staphylococcus aureus enzymes in the extract;

- the optimal composition of the reaction mixture, which makes it possible to obtain an extract without the stage of incubation of the mixture to increase active ribosomes after they have read all the available mRNA in solution;

- the possibility of recording the inhibitory activity of chemical compounds both by recording the fluorescence signal of the reporter protein and luminescence.

The specified technical problem is solved, and the technical result is achieved by the claimed cell-free protein synthesis system based on Staphylococcus aureus cells, obtained by mixing the components of the reaction mixture in the following order and in the following volume ratio (vol.%):

water - 7.85,

100 mM HEPES-KOH pH 8.0 - 5.00,

2% polyethylene glycol 8000 - 4.00,

0.1 mM EDTA pH 8.0 - 0.5,

2 mM dithiothreitol - 0.20,

9.1 mM magnesium acetate - 0.91,

228.5 mM potassium acetate - 5.71,

0.5 mM solution of 20 proteinogenic amino acids - 12.50,

16.7 mM solution of amino acids in an equimolar ratio: arginine, cysteine, tryptophan, methionine, aspartic acid, glutamic acid - 5.99,

0.05% sodium azide - 1.67,

1% protease inhibitor solution Complete - 1.00,

RNase inhibitor RNasine - 0.75,

total tRNA from Escherichia coli – 1.25,

2.11 mM folinic acid - 1.00,

20 mM acetyl phosphate lithium-potassium salt - 4.00,

20 mM phosphoenolpyruvate dipotassium salt - 4.0,

NTP mix: 90 mM ATP, 60 mM CTP, 60 mM GTP, 60 mM UTP, pH 7.0 - 1.33,

T7 polymerase - 2.50;

S30 extract of Staphylococcus aureus RN4220 cells - 35;

plasmid DNA - 4.44.

The composition of the claimed cell-free protein synthesis system is optimal, its change or other than the stated order of adding components leads to deterioration of the system and a decrease in the level of protein synthesis. The inventive composition also makes it possible to obtain an extract without the stage of incubation of the mixture to increase active ribosomes after they have read all the available mRNA in solution.

Also, the technical problem is solved, and the technical result is achieved by the claimed method of protein synthesis in a cell-free protein synthesis system based on Staphylococcus aureus cells, including incubation at a temperature of 37°C of the above-described cell-free protein synthesis system based on Staphylococcus aureus cells.

In addition, the technical problem is solved, and the technical result is achieved by the claimed method for screening protein synthesis inhibitors, including incubation at 37°C of potential protein synthesis inhibitors together with the claimed cell-free protein synthesis system based on Staphylococcus aureus cells, quantitative measurement of the synthesized protein by a luminescent or fluorescent signal radiation and determination of the ability to inhibit protein synthesis by the fall of the light signal.

In the case of using luminescence, luciferin is added to the reaction mixture after incubation of potential inhibitors of protein synthesis together with the inventive cell-free system.

The proposed group of inventions can become a platform for the search for inhibitors of protein synthesis, including selective ones against the bacterium Staphylococcus aureus.

The claimed inventions have the following advantages:

- the number of stages for obtaining a cell-free protein synthesis system is reduced, which reduces costs and labor costs;

- the possibility of using not only luminescence, but also fluorescence in the quantitative determination of protein, which makes it possible to evaluate protein synthesis in dynamics;

- no need to use a commercial preparation of luciferin for luminescence due to its own synthesis of a fluorescent protein.

Also, the cost of obtaining the claimed cell-free system can be reduced by self-synthesis of the recombinant lysostaphin protein without using a commercial preparation of lysostaphin for cell lysis.

The claimed technical solution is illustrated by figures 1 and 2, which show the results of testing the claimed cell-free protein synthesis system of Staphylococcus aureus in the absence and presence of known translational inhibitors:

in fig. 1 - chloramphenicol, where: K ^- - reaction with H ₂ O instead of a reporter plasmid (negative control); K-, luc - reaction with the addition of a non-reporter plasmid (used to exclude the effect of the addition of a non-reporter plasmid on the fluorescence level); K ⁺ , reaction with sfGFP reporter plasmid (signal is taken as 100% level of protein synthesis); 2-100 μM - samples with the addition of chloramphenicol in various concentrations. With an increase in the concentration of chloramphenicol, the synthesis of the GFP protein is suppressed and the level of fluorescence decreases. The measurement was carried out at the final point 8 hours after the start of the reaction. AFU - relative units of fluorescence (Arbitrary Fluorescence Units).

in fig. 2 - tetracycline, where Tet 20 mM - control of the fluorescence of a solution of tetracycline with a concentration of 20 mM; K ^- reaction with H ₂ O instead of reporter plasmid (negative control); K ⁺ , reaction with sfGFP reporter plasmid (signal is taken as 100% level of protein synthesis); CH, chloramphenicol; T, tetracycline; concentration in μM is given in parentheses. With an increase in the concentration of tetracycline, the synthesis of the GFP protein is suppressed and the level of fluorescence decreases. AFU - relative units of fluorescence (Arbitrary Fluorescence Units).

Further, the applicant provides a description of the claimed technical solution.

The claimed inventions are intended for protein synthesis in the cell-free system of Staphylococcus aureus , detection of protein synthesis inhibitors based on the cell-free protein synthesis system based on Staphylococcus aureus cells.

The claimed technical result is generally achieved by the fact that on the basis of cellsStaphylococcus aureusstrain RN4220 an extract is prepared, the components of the reaction mixture are mixed, a protein synthesis reaction is carried out, and the synthesized protein is used, for example, for signal detection in order to evaluate the synthesis efficiency.

All reagents used are commercially available.

The claimed cell-free protein synthesis system, without regard to the order of addition of components, includes, vol.%:

- S30 extract of Staphylococcus aureus RN4220 cells - 35;

- an optimized set of components for carrying out the synthesis reaction - 56.16, which includes:

water - 7.85,

100 mM HEPES-KOH pH 8.0 - 5.00,

2% polyethylene glycol 8000 - 4.00,

0.1 mM EDTA pH 8.0 - 0.5,

2 mM dithiothreitol - 0.20,

9.1 mM magnesium acetate - 0.91,

228.5 mM potassium acetate - 5.71,

0.5 mM solution of 20 proteinogenic amino acids - 12.50,

16.7 mM solution of amino acids in an equimolar ratio: arginine, cysteine, tryptophan, methionine, aspartic acid, glutamic acid - 5.99,

0.05% sodium azide - 1.67,

1% protease inhibitor solution Complete - 1.00,

RNase inhibitor RNasine - 0.75,

total tRNA from Escherichia coli – 1.25,

2.11 mM folinic acid - 1.00,

20 mM acetyl phosphate lithium-potassium salt - 4.00,

NTP mix: 90 mM ATP, 60 mM CTP, 60 mM GTP, 60 mM UTP, pH 7.0 - 1.33,

T7 polymerase - 2.50;

plasmid DNA - 4.44;

ATP regeneration system - 4.4, including:

20 mM phosphoenolpyruvate dipotassium salt - 4.0,

0.18 mM pyruvate kinase - 0.4.

When implementing the invention, the following reagents and equipment were used:

water (>99.9999%, OST 11.029.003-80), HEPES-KOH (≥99.5%, art. H0527, Sigma-Aldrich), polyethylene glycol 8000 (≥99.0%, art. HR2-535, Hampton Research), EDTA (>99.0%, art. H-E5134-0.1, Helicon), dithiothreitol (≥ 99.0%, art. R0861, Thermo Scientific), magnesium acetate (>99.0%, art. Am-O131-0.5, Helicon), potassium acetate (>99.0%, art. P1190-100G, Sigma-Aldrich), 20 proteinogenic amino acids solution (>98%, art. L4461, Promega), amino acids: arginine (≥99.0%, art. 1015420100, Sigma-Aldrich) , cysteine (≥99.0%, art. 1028380025, Sigma-Aldrich), tryptophan (≥99.0%, art. 1083740010, Sigma-Aldrich), methionine (≥99.0%, art. 1057070025, Sigma-Aldrich), aspartic acid (≥ 99.0%, art. 1001260100, Sigma-Aldrich), glutamic acid (≥99.0%, art. 1002910250, Sigma-Aldrich), sodium azide (≥99.5%, art. 71289-5G, Sigma-Aldrich), protease inhibitor Complete ( >95%, art. 11697498001, Roche), RNase inhibitor RNasine (>95%, art. N2111, Promega), total tRNA from Escherichia coli (art. rnamrero, Roche), folinic acid (≥99.0%, art. 47612-250MG, Sigma-Aldrich), acetyl phosphate lithium potassium salt (≥85% art. A0262-5G, Sigma-Aldrich), NTP mix: ATP (≥99.0%, art. R0441, Thermo Scientific), CTP (≥ 99.0%, art. R0451, Thermo Scientific), GTP (≥99.0%, art. R0461, Thermo Scientific), UTP (≥99.0%, art. R0471, Thermo Scientific), T7 polymerase (>95%, art. EP0113, Thermo Scientific), Plasmid DNA (#102634, Addgene), 20 mM phosphoenolpyruvate dipotassium salt (99%, art. 860077-1G, Sigma-Aldrich), Pyruvate kinase (>95%, art. P1506-1KU, Sigma-Aldrich), tablet spectrophotometer-luminometer Varioskan LUX (Thermo Scientific).

The above source components of the claimed system are obtained as follows:

- an extract based on Staphylococcus aureus strain RN4220 cells is prepared using enzymatic lysis with lysostaphin, ultracentrifugation and dialysis, without the stage of additional translation;

- plasmid DNA reporter obtained from transformed cells of Escherichia coli strain DH5a (#102634, Addgene), using the method of alkaline lysis and reprecipitation in ethanol and PEG/CaCl ₂ ;

- an optimized set of components for carrying out the synthesis reaction is obtained by dissolving the components in deionized water.

The light signal is detected by measuring fluorescence or luminescence in a Varioskan LUX plate spectrophotometer (Thermo Scientific).

Thus, the claimed technical solution includes the steps of preparing an extract of Staphylococcus aureus strain RN4220 cells, carrying out a synthesis reaction and signal detection. The extract for carrying out the reactions is made from cells of Staphylococcus aureus strain RN4220 by enzymatic lysis with lysostaphin obtained by a recombinant method. The resulting extract is the basis for a programmable reaction for protein synthesis. To program the system, plasmid DNA encoding a reporter protein (for example, the fluorescent protein sfGFP) is used. The description of the invention contains a protocol for carrying out a synthesis reaction with an optimized set of reagents added to the synthesis reaction, reaction conditions, and conditions for measuring a signal equivalent to the amount of reporter protein in terms of fluorescence level on a plate spectrophotometer.

Further, the applicant provides a method for obtaining the claimed cell-free protein synthesis system based on Staphylococcus aureus cells, the claimed method for protein synthesis based on the cell-free protein synthesis system based on Staphylococcus aureus cells and the claimed method for detecting protein synthesis inhibitors based on the cell-free protein synthesis system based on Staphylococcus aureus cells.

The Staphylococcus aureus RN4220 strain is plated on agar solid medium and incubated for 12 hours at 37°C. A single colony of Staphylococcus aureus cells is seeded in LB medium and the cell mass is grown for 12 hours. Next, the culture is inoculated into fresh medium and the cell mass is grown until the middle of the logarithmic growth phase is reached. The cell mass is then pelleted by centrifugation and the supernatant is removed. The resulting precipitate is resuspended in buffer A (10 mM Tris-Acetate pH 8.2, 14 mM Mg(CH ₃ COO) ₂ , 60 mM KCl, 1 mM DTT) and the cells are washed. The resulting precipitate is stored at -80 °C.

For preparative isolation of reporter plasmid DNA, a combined protocol is used, consisting of a standard protocol for isolation of plasmid DNA and a protocol for isolation of plasmid DNA using CaCl ₂ and PEG 8000. This protocol allows obtaining plasmid DNA without RNA impurities, without the use of RNases and in sufficient concentration (1 μg/μl) for a cell-free system in a short period of time (48 hours). For in vivo expression of the sfGFP reporter protein (superfolded GFP) under the control of the T7 promoter, pJL1-sfGFP plasmid DNA from AddGene (Plasmid #102634) is used, encoding the sfGFP protein sequence, which is used as a standard reporter, highly stable and rapidly folding protein. It is preferable to use plasmid DNA encoding fluorescent proteins - GFP, YFP, CFP, mCherry and their analogues. When using other plasmid DNA, it is necessary to use high copy number vectors in order to increase the yield of plasmid DNA during isolation.

Transformation of cells of Escherichia coli strain DH5a is performed by target plasmid DNA. The cell culture is then grown for 8 hours and inoculated into fresh medium in larger flasks and the cell mass is grown to an OD ₆₀₀ of 0.5. The cells are pelleted by centrifugation and the pellet is resuspended initially in resuspension solution, then in NaOH and SDS lysing solution, after which ammonium acetate is added to the sample to neutralize. The sample is precipitated by centrifugation and isopropyl alcohol is added, incubated and centrifuged. The precipitate is resuspended in TE buffer and ammonium acetate is added, after which the mixture is incubated and centrifuged. Isopropyl alcohol is added to the resulting supernatant, incubated, and then the sample is precipitated by centrifugation. The sample is washed with ethyl alcohol and dried. The resulting pellet is resuspended in highly purified deionized water (mQ). To remove high molecular weight RNA, the sample is treated with CaCl ₂ , after which it is centrifuged, the supernatant is taken, and precipitation is carried out with isopropyl alcohol and potassium acetate, the precipitate is washed with ethyl alcohol and dried. To remove low molecular weight RNA, the pellet is dissolved in TE buffer with PEG/NaCl, mixed and centrifuged. The precipitate is washed with ethyl alcohol and dried. Plasmid DNA is dissolved in a small volume of highly purified deionized water (mQ). For cell-free systems, it is also possible to use plasmid DNA purified using spin column kits - in this case, an additional purification step with phenol-chloroform is necessary to remove RNase.

The extract for the cell-free system is obtained by destroying the cell wall of Staphylococcus aureus with the enzyme lysostaphin. Lysostaphin is obtained by recombinant synthesis in Escherichia coli cells, which reduces the cost of acquiring commercial drugs. The genes encoding the amino acid sequences of lysostaphin without the signal peptide and T7 polymerase are cloned into the pET28A vector for expression in Escherichia coli Bl21. A single colony of Escherichia coli strain BL21 cells transformed with plasmid DNA is seeded from a Petri dish in 25 ml of LB medium with antibiotics corresponding to the resistance marker. The cell mass is grown for 12 h with vigorous stirring at 180 rpm and 37°C. Next, 20 ml of the “overnight” culture is subcultured into 1 liter of LV medium and the cell mass is grown to OD ₆₀₀ 0.5 at 180 rpm and 37 °C. Expression was induced by adding 0.5 mM IPTG and grown with stirring for 4 h at 180 rpm and 37°C. The sedimentation of the cell mass is carried out by centrifugation for 15 minutes at 5400 rpm. Escherichia coli cell mass containing recombinant lysostaphin protein is resuspended in 50 ml of CRB buffer (20 mM Tris-HCl (pH 7.6), 0.5 M NH ₄ Cl) and pelleted by centrifugation for 15 min at 3220 rpm. Escherichia coli cells containing the recombinant lysostaphin protein are thawed in an ice bath and resuspended with 5 ml of cold CRB buffer (20 mM Tris-HCl (pH 7.6), 0.5 M NH ₄ Cl) supplemented with Proteinase Inhibitors Complete (1 tablet per 1 ml) and PMSF (0.5 mm). Cells are lysed using an ultrasonic homogenizer (amplitude 40%, 3 times for 8 minutes, alternated 3 minutes ultrasound on, 5 minutes pause). The lysate is clarified by centrifugation at 25,000 rpm for 30 minutes. The supernatant is transferred to new tubes and re-purified by centrifugation at 45,000 rpm for 45 minutes. Next, the recombinant proteins are purified by metal chelate affinity chromatography. A lysate of Escherichia coli cells containing recombinant lysostaphin protein is added to a column containing Ni-NTA-agarose and washed with 15 ml of wash A buffer (20 mM Tris-HCl (pH 7.6), 1 M NH ₄ Cl), then 15 ml wash B buffer (20 mM Tris-HCl (pH 7.6), 0.5 M NH ₄ Cl, 5 mM imidazole). The protein is washed off the sorbent with 5 ml of elution buffer (20 mM Tris-HCl (pH 7.8), 250 mM NH ₄ Cl, 300 mM imidazole). Additional purification of lysostaphin is carried out using size exclusion chromatography on an NGC Discover FPLC chromatograph (BioRad) and a Superdex 75 10/300 column (GE, UK). Size exclusion chromatography is carried out in a stream of phosphate buffer (25 mM NaH ₂ PO ₄ , 25 mM Na ₂ HPO ₄ , 250 mM NH ₄ Cl) at 4 °C with a loading rate of 0.8 ml/min and 1 ml fractions are collected. Protein separation is followed by the absorption profile of A ₂₈₀ . Fractions are concentrated using Amicon-Ultra concentrators (Merck Millipore, Germany).

Frozen Staphylococcus aureus cells are thawed and buffered with lysostaphin is added, the volume is adjusted to 15 ml to achieve a final extract concentration of 30 mg/ml at the final stage. At this stage, DNases are not used to reduce the viscosity of the solution, since this will lead to hydrolysis of the coding plasmid DNA in the resulting cell-free system. For cell lysis, the resulting mixture is incubated in a water bath. To reduce viscosity and cleave residual DNA, the sample is homogenized with ultrasound at a temperature of 4 °C, after which centrifugation is performed. The resulting cell extract is incubated at 37°C for 60 min in an incubator. The Bradford method is used to determine the concentration of the extract.

To carry out the reaction in the cell-free protein synthesis system of Staphylococcus aureus , sequential addition of reagents is carried out according to an optimized set of components, namely: water, HEPES-KOH pH 8.0, polyethylene glycol 8000, EDTA pH 8.0, dithiothreitol, magnesium acetate, potassium acetate, solution of 20 proteinogenic amino acids, an equimolar mixture of amino acids: arginine, cysteine, tryptophan, methionine, aspartic acid, glutamic acid, sodium azide, protease inhibitor Complete, RNase inhibitor RNasine, total tRNA from Escherichia coli , folinic acid, lithium potassium acetyl phosphate salt, phosphoenolpyruvate dipotassium salt, NTP mix, pyruvate kinase, T7 polymerase, S30 extract of Staphylococcus aureus RN4220 cells, plasmid DNA, separate the reaction mixture in 15 µl in a 384 well plate, incubate at a temperature of 37 ° C and measure the fluorescence or luminescence, obtain the value of the amount synthesized protein. The ability to inhibit protein synthesis by chemical compounds that are potentially inhibitors of protein synthesis, is determined by the fall of the fluorescence signal.

The following are examples of a specific implementation of the invention and the implementation of the purpose, while the applicant implies that the examples given do not limit the intended scope of the claimed technical solution.

Example 1 Preparation of the Claimed Cell-Based Cell-Based Protein Synthesis System Staphylococcus aureus .

1. An extract is obtained from Staphylococcus aureus cells - a component of the claimed system.

Cells of Staphylococcus aureus strain RN4220, which has mutations in the sau 1 hsd R genes, which makes it possible to transform this strain with plasmid DNA from Escherichia coli , are seeded in BHI agar solid medium (Difco) on Petri dishes and incubated for 12 hours at 37 °C . A single colony of Staphylococcus aureus cells is seeded in 20 ml of Lysogeny broth (LB) and the cell mass is grown for 12 h with vigorous stirring at 180 rpm and 37°C. Next, the culture is inoculated into fresh medium in larger flasks at a ratio of 1:100 to fresh LB medium and the cell mass is grown to OD ₆₀₀ = 1.0 (until reaching the middle of the logarithmic growth phase) at 180 rpm and 37 °C. To stop cell growth, the flasks were transferred to an ice bath for 15 min. Then the cell mass is precipitated by centrifugation at 4°C for 15 min at 2500 g and the supernatant is removed. The resulting precipitate is resuspended in 50 ml of buffer A (10 mM Tris-Acetate pH 8.2, 14 mM Mg(CH ₃ COO)2, 60 mM KCl, 1 mM DTT), centrifuged at 4 °C for 15 min at 2500 g, and repeat this procedure three times to remove the remains of the components of the nutrient medium from the solution. The resulting precipitate is weighed and stored at -80 °C.

In the claimed technical solution, the lysis of Staphylococcus aureus cells is obtained by treatment with lysostaphin enzyme recombinantly synthesized in Escherichia coli cells, which reduces the cost of purchasing a commercial drug (for example, L7386-1MG, Sigma).

The genes encoding the amino acid sequences of lysostaphin without the signal peptide and T7 polymerase are cloned into the pET28A vector for expression in Escherichia coli Bl21. A single colony of Escherichia coli strain BL21 cells transformed with plasmid DNA is seeded from a Petri dish in 25 ml of LB medium with antibiotics corresponding to the resistance marker. The cell mass is grown for 12 hours with vigorous stirring at 180 rpm and 37°C. Next, 20 ml of "overnight" culture is inoculated into 1 liter of LV medium and the cell mass is grown to OD ₆₀₀ 0.5 at 180 rpm and 37°C. Expression was induced by adding 0.5 mM IPTG and grown with stirring for 4 h at 180 rpm and 37°C. The sedimentation of the cell mass is carried out by centrifugation for 15 minutes at 5400 rpm. Escherichia coli cell mass containing recombinant lysostaphin protein is resuspended in 50 ml of CRB buffer (20 mM Tris-HCl (pH 7.6), 0.5 M NH ₄ Cl) and pelleted by centrifugation for 15 min at 3220 rpm. Escherichia coli cells containing the recombinant lysostaphin protein are thawed in an ice bath and resuspended with 5 ml of cold CRB buffer (20 mM Tris-HCl (pH 7.6), 0.5 M NH ₄ Cl) supplemented with Proteinase Inhibitors Complete (1 tablet per 1 ml) and PMSF (0.5 mm). Cells are lysed using an ultrasonic homogenizer (amplitude 40%, 3 times for 8 minutes, alternated 3 minutes ultrasound on, 5 minutes pause). The lysate is clarified by centrifugation at 25,000 rpm for 30 minutes. The supernatant is transferred to new tubes and re-purified by centrifugation at 45,000 rpm for 45 minutes. Next, the recombinant proteins are purified by metal chelate affinity chromatography. A lysate of Escherichia coli cells containing recombinant lysostaphin protein is added to a column containing Ni-NTA-agarose and washed with 15 ml of wash A buffer (20 mM Tris-HCl (pH 7.6), 1 M NH ₄ Cl), then 15 ml wash B buffer (20 mM Tris-HCl (pH 7.6), 0.5 M NH ₄ Cl, 5 mM imidazole). The protein is washed off the sorbent with 5 ml of elution buffer (20 mM Tris-HCl (pH 7.8), 250 mM NH ₄ Cl, 300 mM imidazole). Additional purification of lysostaphin is carried out using size exclusion chromatography on an NGC Discover FPLC chromatograph (BioRad) and a Superdex 75 10/300 column (GE, UK). Size exclusion chromatography is carried out in a stream of phosphate buffer (25 mM NaH ₂ PO ₄ , 25 mM Na ₂ HPO ₄ , 250 mM NH ₄ Cl) at 4° C. with a loading rate of 0.8 ml/min and 1 ml fractions are collected. Protein separation is followed by the absorption profile of A ₂₈₀ . Fractions are concentrated using Amicon-Ultra concentrators (Merck Millipore, Germany).

To prepare the extract, prepared cells of Staphylococcus aureus strain RN4220 are used. Frozen cells are thawed and buffer with lysostaphin is added, the volume is adjusted to 5-15 ml to achieve a final extract concentration of 10-30 mg/ml at the final stage. At this stage, DNases are not used to reduce the viscosity of the solution, since this will lead to hydrolysis of the coding plasmid DNA in the resulting cell-free system. For cell lysis, the resulting mixture is incubated in a water bath. To reduce viscosity and cleave residual DNA, the sample is homogenized with ultrasound at a temperature of 4 °C, after which centrifugation is performed. The resulting cell extract is incubated at 37°C for 60 min in an incubator. At this stage, instead of an additional stage of incubation of the lysate to increase the active ribosomes after reading all the available mRNA in the solution and using multicomponent solutions with enzymes and an ATP regeneration system, the extract is dialyzed in the final buffer in dialysis bags with a 14 kDa membrane, centrifuged and frozen in liquid nitrogen. The Bradford method is used to determine the concentration of the extract. Since individual amino acids are poorly soluble, suspensions are used, which are added after thorough mixing to the final volume of the reaction mixture.

2. Get plasmid DNA - a component of the claimed system.

The claimed technical solution for in vivo expression of the sfGFP (superfolded GFP) reporter protein under the control of the T7 promoter uses pJL1-sfGFP plasmid DNA from AddGene (Plasmid #102634), which encodes the sfGFP protein sequence, which is used as a standard reporter, highly stable and rapidly folding protein . For the preparation of a plasmid DNA reporter, a combined protocol is used, consisting of a standard plasmid DNA isolation protocol and a plasmid DNA isolation protocol using CaCl ₂ and PEG 8000. This protocol allows you to obtain plasmid DNA without RNA impurities, without the use of RNases and a concentration of 1 μg / μl for cell-free system in 48 hours.

A single colony of Escherichia coli strain DH5a cells transformed with the pJL1-sfGFP plasmid from a Petri dish with solid LB agar medium is seeded in 25 ml of LB medium with antibiotics corresponding to the resistance marker. The cell mass is grown for 8 h with vigorous stirring at 180 rpm and 37°C. Next, the culture is inoculated into fresh medium in larger flasks at a ratio of 1:100 to fresh LB medium and the cell mass is increased to OD ₆₀₀ 0.5 at 180 rpm and 37°C. To precipitate the cell mass, centrifugation is used at 4 °C for 15 min at 4000 g, the supernatant is removed and the precipitate is processed. The Escherichia coli cell mass is then resuspended in 40 ml of Solution I buffer (5 mM Tris-HCl pH 8.0, 10 mM EDTA, 50 mM glucose) and incubated on ice for 5 minutes. Next, 80 ml of Solution II (0.2 M NaOH, 1% SDS) is added, the solution is mixed, and the incubation is repeated on ice for 5 minutes. Then, 80 ml of chilled (0 °C) 10 M ammonium acetate are added to the sample and the solution is stirred until a homogeneous state. The sample is then incubated on ice for 5 min and pelleted by centrifugation at 4°C for 30 min at 15,000 rpm. The supernatant is filtered through a sterile medical bandage and added to 0.6 volume of isopropyl alcohol, after which it is incubated at room temperature for 10 minutes. Next, centrifugation is performed at 4 °C for 30 min at 15000 rpm, the supernatant is removed and the precipitate is resuspended in 0.8 ml of TE medium. Then 0.2 ml of 10 M ammonium acetate is added and the mixture is incubated on ice for 5 minutes, after which centrifugation is performed at 4 °C for 5 minutes at 15,000 rpm and the supernatant is taken. 0.6 volume of isopropyl alcohol is added to the resulting sample to precipitate plasmid DNA and incubated at room temperature for 10 minutes. Further precipitated by centrifugation at 15,000 rpm, after which the tubes with the sample are inverted over filter paper and the remaining liquid is removed with a pipette. Then the sample is washed with 70% ethanol and dried at 50°C in test tubes with an open lid. The resulting dry precipitate is resuspended in highly purified deionized water and incubated for 15 min at 50°C until the precipitate is completely dissolved. To remove residual RNA, a protocol is used to isolate plasmids with CaCl ₂ and PEG 8000. To do this, high molecular weight RNA is first precipitated by adding CaCl ₂ to 1.4 M and the solution is stirred, then centrifugation is performed at room temperature at 15,000 rpm for 10 min and collect the supernatant. Then, 0.6 volumes of isopropyl alcohol and 0.1 volumes of 3 M potassium acetate are added to the supernatant, the sample is mixed and again precipitated by centrifugation at room temperature for 10 minutes at 15,000 rpm. The resulting precipitate is washed with 70% ethanol and dried in air in an incubator at 37°C. Dissolve the precipitate in TE medium and add 1 volume of PEG/NaCl solution (20% PEG 8000, 500 mM NaCl), mix and centrifuge at room temperature for 10 min at 14000 rpm. Then the precipitate is washed with cold (0°C) 70% ethanol, dried in air in an incubator at 37°C, and this procedure is repeated twice. The thus obtained plasmid DNA is dissolved in a volume of 100 μl of highly purified deionized water and incubated at 37 °C for 15 minutes until complete dissolution. The concentration of plasmid DNA is determined on a nano volume spectrophotometer by absorption at a wavelength of 260 nm. Sample purity and compliance with molecular weight is determined by the agarose gel electrophoresis pattern.

3. Take an optimized set of components for carrying out the synthesis reaction, including an ATP regeneration system from phosphoenolpyruvate dipotassium salt and pyruvate kinase.

4. The above components are mixed at room temperature according to the sequence: water, HEPES-KOH pH 8.0, polyethylene glycol 8000, EDTA pH 8.0, dithiothreitol, magnesium acetate, potassium acetate, a solution of 20 proteinogenic amino acids, a mixture of amino acids in an equimolar ratio : arginine, cysteine, tryptophan, methionine, aspartic acid, glutamic acid, sodium azide, protease inhibitor Complete, RNase inhibitor RNasine, total tRNA from Escherichia coli , folinic acid, acetyl phosphate lithium potassium salt, phosphoenolpyruvate dipotassium salt, NTP mix, pyruvate kinase , T7 polymerase, S30 extract of Staphylococcus aureus RN4220 cells, pJL1-sfGFP plasmid DNA until a homogeneous solution is obtained. Get the claimed cell-free protein synthesis system based on Staphylococcus aureus cells of the claimed composition.

Example 2 Carrying out protein synthesis based on cell-based protein synthesis system Staphylococcus aureus .

The reaction for protein synthesis is carried out in the developed cell-free system. To do this, prepare the reaction mixture in black opaque plates for 384 wells (for example, Grenier), the volume of the wells is 20 μl, the volume of the reaction mixture per well is 15 μl. The list of reagents and the sequence of mixing the reagents are given in Example 1. The quantitative determination of the synthesized protein is carried out by a fluorescent signal, the amount of protein depends on the synthesis time, in this case 8 hours. Fluorescence can be measured both in kinetic mode (within 2-10 hours with measurement every 30 minutes) and in the final point. Fluorescence is measured with a spectrophotometer-luminometer, eg Varioskan LUX (Thermo Scientific), at 485 nm (excitation), 510 nm (emission) in the case of the sfGFP reporter. In addition to recording the synthesis level by fluorescence, it is also possible to change the synthesis level by the luminescence signal. In this case, a reporter plasmid DNA encoding the luciferase sequence is used, and luciferin is added to the reaction mixture to a concentration of 0.1 mM.

The presence of a fluorescence signal with the addition of plasmid DNA that exceeds the signal without the addition of plasmid DNA indicates the presence of the fluorescent sfGFP protein and, thus, successful protein synthesis using a cell-free protein synthesis system based on Staphylococcus aureus cells.

Example 3 Determination of the Ability of Chloramphenicol and Tetracycline to Inhibit Protein Synthesis in a Cell-Based Cell-Based Protein Synthesis System Staphylococcus aureus .

The synthesis of the fluorescent GFP protein is carried out in the developed cell-free system in black opaque plates for 384 wells (Grenier), the volume of the wells is 20 μl and the volume of the reaction mixture in the well is 15 μl. The sequence of steps for preparing tablets in is given in Example 2. In different tablets in three repetitions, the known inhibitors of protein synthesis - chloramphenicol (dissolved in DMSO) and tetracycline (dissolved in mQ) - were added to the reaction mixture at various concentrations. For the sample dissolved in DMSO, in order to exclude the influence of the solvent on the translation process, DMSO was also added to the control sample (K+) to a concentration of 1.66%. The volume of the reaction mixture for the reaction in the 384 well plate was 15 μl per well (well volume 20 μl). Fluorescence was measured at the endpoint at 30°C after 8 hours. Fluorescence was measured using black opaque 384-well plates (Grenier, USA). Fluorescence was measured at 485 nm (excitation), 510 nm (emission) (Varioskan LUX, Thermo).

The results are presented in figures 1 and 2, which shows the levels of protein synthesis in the claimed cell-free translation system of Staphylococcus aureus in the absence and presence of these known translational inhibitors at various concentrations. As a control, a reaction with H ₂ O instead of reporter plasmid DNA (negative control) and a reaction with the addition of non-reporter plasmid DNA were carried out simultaneously in order to exclude the influence of the addition of non-reporter plasmid DNA on the level of fluorescence. The reaction with reporter plasmid DNA sfGFP without the addition of inhibitors is taken as a 100% synthesis level. Taking the intensity of the fluorescence signal as a 100% level of protein synthesis for the well with the sfGFP reporter plasmid, it can be seen that when the reaction mixture is incubated with chloramphenicol at a concentration of 2 μM, 3 μM, 6 μM, 15 μM, 25 μM, 100 μM, the level of protein synthesis decreases by 15 %, 15%, 22%, 39%, 51% and 87% respectively. And when the reaction mixture is incubated with tetracycline at a concentration of 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, the level of protein synthesis decreases by 33%, 74%, 94%, 97%, 97% 97%.

Thus, carrying out manipulations in accordance with the description above makes it possible to achieve the stated goal - to obtain a cell-free protein synthesis system based on Staphylococcus aureus cells, to carry out protein synthesis, and to identify inhibitors of protein synthesis based on it. The examples of implementation of the claimed invention show the successful creation of a cell-free protein synthesis system based on Staphylococcus aureus cells, carrying out protein synthesis and identifying protein synthesis inhibitors with its use, which demonstrates the usefulness of applying the claimed inventions in pharmacology, for example, to search for protein synthesis inhibitors, while using standard equipment and materials.

Based on the foregoing, it can be concluded that the applicant has achieved the claimed technical result, namely, the arsenal of means for the indicated purpose has been expanded by creating a cell-free protein synthesis system based on Staphylococcus aureus cells, protein synthesis has been carried out and inhibitors of protein synthesis based on it have been identified, while achieving:

- the possibility of protein synthesis of gram-positive bacteria Staphylococcus aureus in the natural genetic and metabolic environment;

- the possibility of obtaining an extract without the stage of incubation of the mixture to increase active ribosomes after reading all the available mRNA in solution;

- the possibility of synthesizing recombinant lysostaphin protein without using a commercial preparation of lysostaphin for cell lysis;

- the possibility of recording the inhibitory activity of chemical compounds both by recording the fluorescence signal of the reporter protein and luminescence.

In addition, the proposed group of inventions can become a platform for the search for inhibitors of protein synthesis in the bacterium Staphylococcus aureus .

The claimed technical solution satisfies the criterion of "novelty" for inventions, since when determining the level of technology, no means were identified that have features that are identical (that is, they match in the function they perform and the form of execution of these features) to all the features listed in the claims of the alleged invention , including the characteristics of the destination.

The claimed technical solution satisfies the criterion of "inventive step", since it is not obvious to a specialist in this field of technology - from the studied prior art, the applicant has not identified technical solutions that coincide in technical essence with the proposed solution, and the influence of distinctive features on the obtained technical result has not been established .

The claimed technical solution complies with the "industrial applicability" patentability requirement for inventions, as it can be manufactured using known materials and equipment.

Claims (70)

1. Cell-free protein synthesis system based on Staphylococcus aureus cells, obtained by mixing the components of the reaction mixture in the following order and in the following volume ratio, vol.%: water - 7.85, 100 mM HEPES-KOH pH 8.0 - 5.00, 2% polyethylene glycol 8000 - 4.00, 0.1 mM EDTA pH 8.0 - 0.5, 2 mM dithiothreitol - 0.20, 9.1 mM magnesium acetate - 0.91, 228.5 mM potassium acetate - 5.71, 0.5 mM solution of 20 proteinogenic amino acids - 12.50, 16.7 mM solution of amino acids in an equimolar ratio: arginine, cysteine, tryptophan, methionine, aspartic acid, glutamic acid - 5.99, 0.05% sodium azide - 1.67, 1% protease inhibitor solution Complete - 1.00, RNase inhibitor RNasine - 0.75, total tRNA from Escherichia coli – 1.25, 2.11 mM folinic acid - 1.00, 20 mM acetyl phosphate lithium-potassium salt - 4.00, 20 mM phosphoenolpyruvate dipotassium salt - 4.0, 0.18 mM pyruvate kinase - 0.4, NTP mix: 90 mM ATP, 60 mM CTP, 60 mM GTP, 60 mM UTP, pH 7.0 - 1.33, T7 polymerase - 2.50; S30 extract of Staphylococcus aureus RN4220 cells - 35; plasmid DNA - 4.44. 2. Cell-free protein synthesis system based on Staphylococcus aureus cells according to claim 1, characterized in that in the case of quantitative determination of the synthesized protein by fluorescence, pJL1-sfGFP is used as plasmid DNA. 3. A cell-free protein synthesis system based on Staphylococcus aureus cells according to claim 1, characterized in that in the case of quantitative determination of the synthesized protein by the luminescence method, pT7luc is used as plasmid DNA. 4. A method for protein synthesis in a cell-free protein synthesis system based on Staphylococcus aureus cells, including incubation at a temperature of 37 ° C of a cell-free protein synthesis system based on Staphylococcus aureus cells, obtained by mixing the components of the reaction mixture in the following order and in the following volume ratio, vol. %: water - 7.85, 100 mM HEPES-KOH pH 8.0 - 5.00, 2% polyethylene glycol 8000 - 4.00, 0.1 mM EDTA pH 8.0 - 0.5, 2 mM dithiothreitol - 0.20, 9.1 mM magnesium acetate - 0.91, 228.5 mM potassium acetate - 5.71, 0.5 mM solution of 20 proteinogenic amino acids - 12.50, 16.7 mM solution of amino acids in an equimolar ratio: arginine, cysteine, tryptophan, methionine, aspartic acid, glutamic acid - 5.99, 0.05% sodium azide - 1.67, 1% protease inhibitor solution Complete - 1.00, RNase inhibitor RNasine - 0.75, total tRNA from Escherichia coli – 1.25, 2.11 mM folinic acid - 1.00, 20 mM acetyl phosphate lithium-potassium salt - 4.00, 20 mM phosphoenolpyruvate dipotassium salt - 4.0, 0.18 mM pyruvate kinase - 0.4, NTP mix: 90 mM ATP, 60 mM CTP, 60 mM GTP, 60 mM UTP, pH 7.0 - 1.33, T7 polymerase - 2.50; S30 extract of Staphylococcus aureus RN4220 cells - 35; plasmid DNA - 4.44. 5. A method for detecting inhibitors of protein synthesis, including incubation at 37 °C of potential inhibitors of protein synthesis together with a cell-free protein synthesis system based on Staphylococcus aureus cells, obtained by mixing the components of the reaction mixture in the following order and in the following volume ratio, vol.%: water - 7.85, 100 mM HEPES-KOH pH 8.0 - 5.00, 2% polyethylene glycol 8000 - 4.00, 0.1 mM EDTA pH 8.0 - 0.5, 2 mM dithiothreitol - 0.20, 9.1 mM magnesium acetate - 0.91, 228.5 mM potassium acetate - 5.71, 0.5 mM solution of 20 proteinogenic amino acids - 12.50, 16.7 mM solution of amino acids in an equimolar ratio: arginine, cysteine, tryptophan, methionine, aspartic acid, glutamic acid - 5.99, 0.05% sodium azide - 1.67, 1% protease inhibitor solution Complete - 1.00, RNase inhibitor RNasine - 0.75, total tRNA from Escherichia coli – 1.25, 2.11 mM folinic acid - 1.00, 20 mM acetyl phosphate lithium-potassium salt - 4.00, 20 mM phosphoenolpyruvate dipotassium salt - 4.0, 0.18 mM pyruvate kinase - 0.4, NTP mix: 90 mM ATP, 60 mM CTP, 60 mM GTP, 60 mM UTP, pH 7.0 - 1.33, T7 polymerase - 2.50; S30 extract of Staphylococcus aureus RN4220 cells - 35; plasmid DNA - 4.44, quantitative measurement of the synthesized protein by the signal of luminescent or fluorescent radiation and determination of the ability to inhibit protein synthesis by the fall of the light signal. 6. A method for detecting protein synthesis inhibitors according to claim 5, characterized in that in the case of quantitative measurement of the synthesized protein by a luminescent signal, after incubation of potential inhibitors of protein synthesis, together with a cell-free protein synthesis system based on Staphylococcus aureus cells, luciferin is added to the reaction mixture.