CN115851558B - Novel prokaryotic cell-free protein synthesis system and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a prokaryotic cell-free protein synthesis system and application thereof. The invention develops and establishes a cell-free protein synthesis system by taking rhodococcus (Rhodococcus rhodochrous) as chassis bacteria through optimizing the preparation process of cell culture and extract and CFPS reaction system and reaction conditions; the expression quantity of the target protein is improved by about 10 times compared with that before optimization by using the system. The rhodococcus CFPS system developed by the invention not only expands the current cell-free protein synthesis (CFPS) system library, but also expands the application of the rhodococcus system in the field of synthetic biology, such as rapid protein synthesis, genetic circuit prototype and metabolic pathway construction, and can rapidly and efficiently produce valuable chemicals, materials and the like, and the establishment of the rhodococcus cell-free protein synthesis system also provides possibility for a functional enzyme efficient screening platform and accelerates high-performance enzyme screening.

Description

Novel prokaryotic cell-free protein synthesis system and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a novel prokaryotic cell-free protein synthesis system, which comprises application of the system and a production method.

Background

Cell-free protein synthesis (CFPS) systems use crude cell extracts instead of whole cells to accomplish in vitro transcription and translation are powerful platforms for protein synthesis and biological applications. Due to the openness of cell-free systems, CFPS reactions have many advantages, such as ease of handling, high yields and tolerance to toxic products. CFPS technology has been applied to the production of a variety of proteins, including membrane proteins, therapeutic proteins, metalloenzymes, and unnatural amino acid modified proteins.

CFPS systems were developed from different prokaryotic and eukaryotic or organisms (e.g., e.coli, yeast, wheat germ, and mammalian cells). Although these mature CFPS systems are widely used, they each have advantages and disadvantages. For example, the overall productivity of E.coli CFPS is currently highest (expressed protein about 1000. Mu.g/mL) in all reported cell-free systems; however, cell extracts based on E.coli lack post-translational modification mechanisms (e.g.glycosylation) and therefore they are not suitable for expressing eukaryotic proteins.

On the other hand, the preparation steps of eukaryote-derived CFPS systems are laborious and their protein yields are often low (< 50 μg/mL). Recently, interest in CFPS technology has driven the development of new cell-free systems, including streptomyces, bacillus subtilis, pseudomonas putida, vibrio natrii, and the like. The development of new CFPS systems will not only extend the kits for in vitro protein synthesis, but more importantly, these systems can better mimic the cell's endogenous environment to achieve high quality protein expression (e.g., enhanced solubility and post-translational modification). Thus, the development of CFPS systems derived from non-model chassis microorganisms is increasingly attractive for specific applications in the fields of synthetic biology and biotechnology.

Rhodococcus is an aerobic, spore-free gram-positive bacterium that is widely distributed in a variety of nutritional conditions. One notable feature is that rhodococcus strains exhibit diverse metabolic activities that enable them to degrade and convert different types of organic contaminants, such as short chains, long chains, halogenated hydrocarbons and numerous aromatic hydrocarbons. Thus, they have been successfully applied to bioremediation of contaminated soil, water and air. This factor also makes rhodococcus an ideal platform for industrial waste or lignocellulosic biomass feedstocks to produce valuable chemicals, such as biofuels and carboxylic acids. Rhodococcus has been widely used in industrial production of nitrile hydratase (NHase), nitrilase and amidase, which catalyze the conversion of acrylonitrile to acrylamide and ammonium acrylate or aromatic and heterocyclic amides to the corresponding acids. Thus, rhodococcus has considerable significance in pharmaceutical, environmental and industrial fields, and is considered as one of the microbial platforms for manufacturing chemicals.

There is currently no efficient system for cell-free protein synthesis by rhodococcus. The establishment of the rhodococcus cell-free protein synthesis system can expand the current CFPS library, can also utilize the rhodococcus cell-free protein expression system to build a high-flux enzyme library screening platform so as to accelerate high-performance enzyme screening, and can expand the application of the rhodococcus cell-free protein synthesis system in the field of synthetic biology. Such as rapid protein synthesis, genetic circuit prototypes, and metabolic pathway construction, and the rapid and efficient production of valuable chemicals and materials. How to build an efficient system for cell-free protein synthesis in rhodococcus is a problem to be solved.

Disclosure of Invention

Cell-free protein synthesis (CFPS) systems are powerful platforms for protein synthesis and biological applications, and rhodococcus has considerable significance in pharmaceutical, environmental and industrial fields, and are considered to be one of the microbial platforms for the manufacture of chemicals. Although some CFPS systems have been reported, the protein expression level is low, which is difficult to be practically used, and these systems are not necessarily suitable for expression of rhodococcus genes. There is no rhodococcus CFPS system available at present, which limits the use of rhodococcus. The rhodococcus has thicker cell walls, and the requirements on nutrient components of the culture medium in the protein expression process are high, which all increase the difficulty for developing the rhodococcus CFPS system. Through research and study, the invention aims to develop a CFPS system based on rhodococcus cell extract, and improve the protein expression level so as to expand the current CFPS library and further expand the application of rhodococcus in the field of synthetic biology. Such as rapid protein synthesis, genetic circuit prototypes, and metabolic pathway construction, and the rapid and efficient production of valuable chemicals and materials.

The invention firstly provides a preparation method of rhodococcus cell extract for a cell-free protein synthesis system, which comprises the following steps:

(1) And (3) culturing the rhodococcus, namely fermenting and culturing the rhodococcus by adopting a fermentation medium, wherein the fermentation medium comprises the following components: 10-50 g of glucose, 1-10 g of yeast powder, 1-8 g of peptone, 1-8 g of malt extract, 0.1-1 g of dipotassium hydrogen phosphate, 0.1-1 g of monopotassium phosphate, 0.5-3 g of magnesium sulfate, 0.5-3 g of urea and water to a constant volume of 1L, and the pH is 7.2, and the temperature is 115 ℃ for 20 min.

(2) And (3) thallus collection: when fermenting and culturing to OD of thallus ₆₀₀ Collecting thalli in 1.9-3.1; and adjusting the concentration of the bacterial cells to be 0.6-0.8 g/mL;

(3) And (3) thallus crushing: the method comprises the steps of adopting ultrasonic bacteria breaking, wherein the ultrasonic conditions are that the power is 50%, the ultrasonic is 2 s, the interval is 6 s, and the total time is 6-8 min, so as to obtain a cell extract; wherein glycerol is added before sonication or dithiothreitol is added after sonication.

Preferably, in step (3), 10% glycerol is added before sonication, more preferably 10-20ul 1m DTT per 1 mL broth.

The invention provides a rhodococcus cell-free protein synthesis system, which comprises the following components:

(1) PEG8000 addition: 1-2% w/v;

(2) Addition amount of rhodococcus cell extract: 25-33% (v/v);

(3) The addition amount of the DNA template: 12-18 ng/uL plasmid;

(4) The addition amount of the RNase inhibitor: 0.1 to 0.2U/uL;

(5) Addition amount of T7RNA polymerase: 0.6-0.8U/uL;

and ATP, GTP, UTP and CTP, folinic acid, 20 standard amino acids, nicotinamide adenine dinucleotide; coenzyme A; spermidine; putrescine; sodium oxalate; potassium glutamate; ammonium glutamate; magnesium glutamate; HEPES; phosphoenolpyruvic acid.

Preferably, the following components are added to a final concentration: ATP 1.2 mM; GTP, UTP and CTP are each 0.80-0.90 mM;30-40 μg/mL folinic acid; 2-6 ng/. Mu.L plasmid; 2.5-3.5 mM of 20 standard amino acids each; 0.30-0.36 mM nicotinamide adenine dinucleotide; 0.25-0.30 mM coenzyme A; 1.0-2.0. 2.0 mM spermidine; 0.5-1.5 mM putrescine; 2-6 mM sodium oxalate; 280-300 mM potassium glutamate; 8-12 mM ammonium glutamate; 4-8 mM magnesium glutamate; 50-60 mM HEPES pH 7.2; 65-70 mM phosphoenolpyruvic acid.

More preferably, the ingredients are added in the final concentrations of: ATP 1.2 mM; GTP, UTP and CTP each 0.85 mM; 34. mu g/mL folinic acid; 4 ng/. Mu.L plasmid; 0.2 U/uL T7RNA polymerase; 0.1 U/uL RNase inhibitors; each 3 mM of the 20 standard amino acids; 0.33 mM nicotinamide adenine dinucleotide; 0.27 mM coenzyme A;1.5 mM spermidine; 1 mM putrescine; sodium 4 mM oxalate; 290 mM potassium glutamate; 10 mM ammonium glutamate; 6 mM magnesium glutamate; 57 mM HEPES pH 7.2;67 mM phosphoenolpyruvate and 33% Rhodococcus cell extract.

Further preferably, the rhodococcus cell extract is prepared by the method; the pH of the system (i.e., the reaction system) is 7.0 to 7.4.

The invention further provides a method for synthesizing the rhodococcus cell-free protein, which is characterized in that the system is adopted to react for 20-44h at 15-20 ℃.

The invention also provides the use of said system, or said method, in cell-free protein synthesis.

More specifically, it is used for rapid protein synthesis, rapid construction of enzyme mutation libraries, genetic circuit prototypes or metabolic pathway construction.

The invention develops and establishes a cell-free protein synthesis system taking rhodococcus (rhodococcus) as chassis fungus by optimizing the preparation process of cell culture and extracts thereof and a CFPS reaction system and reaction conditions; by using the system, the expression quantity of the target protein can reach more than 300 mu g/mL. The rhodococcus CFPS system developed by the invention is an initial in the industry, not only expands the current CFPS library, but also expands the application of rhodococcus in the field of synthetic biology, such as rapid protein synthesis, genetic circuit prototype and metabolic pathway construction, and rapid and efficient production of valuable chemicals, materials and the like.

Drawings

FIG. 1 Rhodococcus CFPS expression plasmid PJL1-eGFP.

FIG. 2 CFPS system developed under a gel tester. And (3) injection: an experimental group A; b negative control.

FIG. 3 CFPS system developed under a gel tester. And (3) injection: a pre-optimized CFPS system, B negative control, C post-optimized CFPS system, D negative control (where a and B are a and B in fig. 2, in fig. 3 for visual comparison).

FIG. 4 shows comparison of eGFP expression levels before and after optimization.

Detailed Description

The process according to the invention is further illustrated by the following examples. The experimental method in which specific conditions are not specified in examples can be generally performed according to conditions in a routine experiment in the field of molecular biology or according to instructions of commercial manufacturers such as plasmids and strains. The present invention may be better understood and appreciated by those skilled in the art by reference to the examples.

Example 1: recombinant plasmid construction of reporter gene eGFP

The cell-free protein synthesis requires a target gene to verify whether the system is available, and the green fluorescent protein (eGFP) is a green fluorescent protein, so that the concentration of the synthesized protein can be conveniently and rapidly detected, and the CFPS system can be evaluated.

Primers were designed based on eGFP sequence information, and eGFP was amplified by PCR using KOD high-fidelity polymerase with plasmid pET28a containing eGFP as a template. The PCR procedure was: 94 ℃ 120s,98 ℃ 10s,68 ℃ 30s,68 ℃ 10s,18 cycles, purifying the PCR product, digesting the purified product and the expression vector PJL1 by restriction enzymes, recovering and then carrying out ligation reaction. The ligation product was transformed into E.coli DH 5. Alpha. And plated on LB plates containing 50. Mu.g/mL kanamycin for selection. Positive transformants were selected, plasmids were extracted and confirmed by sequencing, and the results showed correct columns, and recombinant plasmids were obtained as shown in fig. 1.

Example 2: optimized expression eGFP of rhodococcus CFPS system

1. Conventional PANOx-SP System for expression of eGFP by Rhodococcus CFPS

To initially verify the expression level of the rhodococcus cell-free protein expression system, the verification was performed by the following experiments, in particular as follows:

1. rhodococcus cell culture and preparation of cell extract thereof: rhodococcus was activated on a solid LB medium, and then inoculated with activated monoclonal into 40 ml of LB liquid medium, followed by culturing at 28℃and 200 rpm overnight. Inoculating overnight culture broth to 400 ml 2×YInitial OD was controlled in TP Medium ₆₀₀ Culturing at 28 ℃ to OD (optical density) at 0.1-0.5 ₆₀₀ Collecting thalli at 3.5-4.0, centrifuging for 20 min at 4 ℃ with 10000 g, discarding supernatant, and adding 25 ml of S30 buffer (10 mM Tris (OAc) and 1.4 mM Mg (OAc) ₂ The cells were rinsed 2 times with 60 mM K (OAc) and 2 mM DTT, and the cells were collected and stored at-80℃for use. Weighing 1g, adding 1 ml precooled S30 buffer to resuspend cells, ultrasonic breaking (phi 6 ultrasonic probe, 50% power, ultrasonic 2S, stop 6S, total time 4 min), 10000 rpm, centrifuging at 4 ℃ for 30 min. The supernatant, the cell extract, was dispensed into 1.5 mL EP tubes and stored at-80℃for further use.

2. The rhodococcus CFPS reaction system is built according to a conventional PANOx-SP system, and the specific components are as follows: 1.2 mM ATP; GTP, UTP and CTP each 0.85 mM; 34. mu g/mL folinic acid; 4. 4 ng/. Mu.L plasmid, 0.2U/uL T7RNA polymerase, 0.1U/uL RNase inhibitor; each 3 mM of the 20 standard amino acids; 0.33 mM Nicotinamide Adenine Dinucleotide (NAD); 0.27 mM coenzyme A (CoA); 1.5 mM spermidine; 1 mM putrescine; sodium 4 mM oxalate; 290 mM potassium glutamate; 10 mM ammonium glutamate; 6 mM magnesium glutamate; 57 mM HEPES pH 7.2;67 mM phosphoenolpyruvate (PEP) and 33% of cell extract, incubated at 30℃for 20 h, and protein expression was observed in real time.

3. Qualitative and quantitative detection of eGFP: firstly, observing whether a reaction system shows green or not by using a nucleic acid gel irradiation instrument, and qualitatively judging whether eGFP is expressed or not; then, 10 μl of the reaction system is taken, 90 μl of 50mM HEPES is added, and after mixing, 80 μl is taken to 96-well black ELISA plates, the absorbance of eGFP protein is detected by ELISA, a standard curve of the relationship between the fluorescence value and the protein concentration is established by using eGFP standard, and the eGFP protein concentration in the CFPS reaction is quantified.

The experimental result is shown in figure 2, and the reaction system has little green fluorescent protein expression under the nucleic acid gel irradiation instrument; the concentration is calculated by detection of an enzyme label instrument, and the expression level is low as shown in the table 1 and is only 30 ug/mL.

Table 1: conventional PANOx-SP system rhodococcus CFPS protein expression condition

2. Rhodococcus CFPS system optimization

The rhodococcus CFPS reaction system constructed according to the conventional PANOx-SP system has low protein expression level, and cannot meet the requirement of a cell-free system on protein evaluation. In order to increase the availability of rhodococcus CFPS, key factors in cell-free systems are: cell culture, preparation process of cell extract, reaction system and reaction condition are optimized, and protein expression level of cell-free system is raised. The method comprises the following steps:

1. and (3) optimizing a reaction system and reaction conditions:

(1) PEG8000 addition: since cell-free extracts are much less dense (20-30 fold) than the cytoplasmic environment within the cell, the addition of crowding agents is required to mimic the intracellular environment. A crowding agent commonly used in CFPS is polyethylene glycol (PEG), and PEG of various molecular weights is added to CFPS, but its characteristics vary with the molecular weight. PEG8000 is often used for simulating macromolecular congestion, PEG8000 is added into a CFPS system, other conditions are unchanged, example 2 is adopted, eGFP expression level is obviously improved, and specific results are shown in Table 2. The results show that PEG8000 is an important factor affecting the expression level of the rhodococcus CFPS protein, but the concentration of PEG8000 increases, so that the effect on the protein expression level is relatively large, and the most effective concentration range is 1-2% w/v from the prior data.

Table 2: PEG8000 addition amount optimizing protein expression condition

(2) Cell extract addition: the cell extract contains a series of important substances such as RNA polymerase, ribosome, amide-tRNA synthetase, translation initiation and elongation factors and the like which are required for protein synthesis, and the addition amount of the cell extract directly influences the concentration of the active ingredients. The present invention optimizes the amount of the cell extract to be added, and other conditions are the same as those in the first aspect. The specific results are shown in Table 3, and it was found that the addition amount of the cell extract was not as large as possible, and the optimum addition amount was determined to be 25 to 33% (v/v).

Table 3: cell extract addition optimizing protein expression

(3) Addition amount of DNA template (PJL 1-eGFP): cell-free synthesis reactions are a very complex system, and the amount of DNA template is very important, determining the expression level of the later protein. The invention is optimized for the final concentration of the DNA template, and other conditions are the same as the first point. Specific results are shown in Table 4, the expression level tended to increase with increasing amount of DNA template, but the protein expression level did not increase continuously to a certain concentration, which suggests that there may be other restriction factors, and we determined that the optimal template concentration was 12-18 ng/uL plasmid based on this system.

Table 4: optimizing protein expression in final concentration of template

(4) The addition amount of the RNase inhibitor: an rnase inhibitor is a protein that is very effective in inhibiting rnase. In the whole cell-free synthesis reaction, the added template is DNA, mRNA is required to be transcribed in the reaction, but if RNase exists in the reaction, mRNA can be rapidly degraded, and the later protein can not be synthesized; since rnases are widely present in nature, contamination with rnases in experiments is unavoidable, and it is necessary to add an rnase inhibitor to the reaction system, but since the rnase inhibitor is very expensive, and the addition of excessive amounts may inhibit the synthesis of proteins in the cell system. The concentration of the RNase inhibitor is thus optimized in the present invention, with the other conditions being the same as in the first point. Specific results are shown in Table 5, and as the concentration of the RNase inhibitor increases, protein expression is improved, but not significantly, and the addition of excessive amount does inhibit protein synthesis and reduces protein expression level; based on the above results, the optimum concentration of RNase inhibitor to be added is 0.1 to 0.2U/uL.

Table 5: optimizing protein expression at final concentration of RNase inhibitor

(5) Addition amount of T7RNA polymerase: since the promoter on the PJL1 vector is a T7 promoter, T7RNA polymerase is required in the protein synthesis process, and the rhodococcus cell extract is free of T7RNA polymerase, so that additional addition is required in the system. The amount of T7RNA polymerase affects transcription efficiency. The invention optimizes the addition amount of T7RNA polymerase, sets 4 different concentrations and has other conditions similar to the first point. The specific results are shown in Table 6, and the results show that the protein expression level is gradually increased with the increase of the T7RNA polymerase concentration, and the T7RNA polymerase addition concentration is 0.6-0.8U/uL based on the experiment.

Table 6: optimizing protein expression for final concentration of T7RNA polymerase

(6) Reaction temperature: the optimal protein expression temperature of different chassis bacteria is different, and the optimal expression temperature of different enzymes is also different. The invention optimizes the reaction temperature, and other conditions are the same as the first point, and the experiment sets 4 temperatures in total. The result shows that the low-temperature expression of the protein is higher than the high-temperature expression yield, and experiments prove that the optimal temperature range of expressing eGFP by the rhodococcus CFPS is 15-20 ℃.

Table 7: optimizing protein expression at reaction temperature

(7) Reaction time: the protein expression level was gradually increased with the increase of the reaction time, and the protein expression level was compared with the different reaction time under the conditions of example 2, and as a result, it was found that the protein concentration of reaction 44h was not significantly increased as compared with that of reaction 20 h, and based on the results of this experiment, a suitable reaction time was determined to be about 20 h for the purpose of improving the efficiency.

Table 8: optimizing protein expression profile for reaction time

2. Cell extract preparation process optimization:

cell extracts contain a number of important substances such as RNA polymerase, ribosome, amide-tRNA synthetase, translation initiation and elongation factors, which are required for protein synthesis, and thus cell culture and preparation of cell extracts are particularly important. The invention is applied to culture medium and bacteria collection OD ₆₀₀ The ultrasonic time, the concentration of bacteria before ultrasonic and the addition of protein protectant are optimized systematically.

(1) Culture medium: during cell growth, the composition and content of the culture medium have an important effect on cell growth, which in turn affects the quality of the cell extract prepared in the later cell-free synthesis. The bacterial cell extract CFPS cell culture medium reported so far is mainly 2×ytp medium. The present invention co-optimizes 3 media, 2 XYTP (Table 9-2), TB (Table 9-3) and fermentation (Table 9-1), with the other conditions being the same as the first point. The results are shown in Table 10, wherein the difference between the protein expression level of TB medium and 2 XYTP is not obvious, and the protein expression level of fermentation medium is obviously improved, which is probably the effect of salt ions, urea and malt extract in the medium, and the condition medium is determined to be the fermentation medium according to the experimental result.

Table 9-1: fermentation medium formula

Table 9-2:2 XYTP culture medium formula

Table 9-3: TB medium formula

Table 10: optimizing protein expression in culture medium

(2) Bacterial collection OD ₆₀₀ : in the cell growth process, the cell growth condition can influence the content of ribosomes in cells, thereby influencing the synthesis rate and the expression quantity of the protein in the later period. The growth state of cells in the mid-log phase is best, wherein a large number of important substances such as ribosomes, amino acid elongation factors and the like required for protein synthesis are present, and one important index for measuring the growth state of cells is OD ₆₀₀ . The invention is applied to the bacterial recovery OD ₆₀₀ The optimization is performed, and other conditions are the same as the first point. The results are shown in Table 11, in which the OD is shown ₆₀₀ The protein expression level is obviously higher than 3.8-5.6 when the protein is 1.9-3.1, and the result shows that the OD of the bacteria is obtained ₆₀₀ Is an important factor, and attention should be paid to follow-up OD during culture ₆₀₀ Preventing overgrowth of bacterial cells, and determining the suitable bacterial recovery OD according to the experimental result ₆₀₀ 1.9 to 3.1.

Table 11: bacterial OD optimized protein expression condition

(3) Ultrasonic time: how to release the substances required for the intracellular protein synthesis of rhodococcus is an important influencing factor, so the energy parameters during ultrasonic disruption are extremely critical. Rhodococcus belongs to gram-positive bacteria, has thicker cell walls, and can cause incomplete release of substances required in cells to influence the final result when the rhodococcus cannot be completely broken if ultrasonic energy is insufficient, but can destroy substances required for synthesizing a series of proteins such as released ribosomes, energy and the like if the ultrasonic energy is too high. The invention optimizes the ultrasonic time, and other conditions are the same as the first point. The results are shown in Table 12, in which: before 6min, the protein expression amount is higher and higher along with the extension of the ultrasonic time, after 6min, the ultrasonic time is prolonged, and the protein expression amount is slightly reduced, which is consistent with theoretical analysis, and the best ultrasonic condition is determined to be 50% of power, 2% of ultrasonic waves s and 6-s intervals, which are 6-8 min.

Table 12: ultrasound time optimized protein expression profile

(4) Concentration of pre-ultrasound thallus: in addition to ultrasonic energy, the concentration of bacterial sludge prior to ultrasound also directly affects the ultrasound effect. The invention optimizes the concentration of the bacteria before ultrasonic treatment, and other conditions are the same as the first point. The results are shown in Table 13, wherein the cell concentration is slightly higher than 1g/mL from 0.6 to 0.8 g/mL, but the improvement is not significant, but the optimum concentration is determined to be 0.6 to 0.8 g/mL for cost saving.

Table 13: protein expression condition is optimized to fungus mud concentration before supersound

(5) Protein protectant: the ultrasound process can destroy the structure of enzymes related to protein synthesis, resulting in reduced or complete inactivation of enzyme activity, thereby affecting protein expression. In order to stabilize the protein structure and reduce the loss of enzyme activity, glycerol is added before the ultrasonic treatment or dithiothreitol is added after the ultrasonic treatment, and other conditions are the same as the first point. The results are shown in Table 14, wherein the 15 uL 1M DTT protein expression level is not obviously improved after 1 mL bacterial liquid is subjected to ultrasonic treatment, the DTT in the system is possibly sufficient, the 100 uL glycerol protein expression level is obviously improved before ultrasonic treatment, the glycerol possibly plays a role in protecting the protein, and 100 uL glycerol is added into 1 mL bacterial liquid before ultrasonic treatment.

Table 14: protein expression is optimized by adding protein protectant

Example 3 validation of eGFP expression levels Using optimized Rhodococcus CFPS

(1) The optimized rhodococcus cell culture and extract preparation conditions: rhodococcus was activated on a solid LB medium, and then inoculated with activated monoclonal into 40 ml of LB liquid medium, followed by culturing at 28℃and 200 rpm overnight. Inoculating overnight culture broth into 400 ml fermentation medium (Table 9-1), and controlling initial OD ₆₀₀ Culturing at 28 ℃ to OD (optical density) at 0.1-0.5 ₆₀₀ 1.9 to 3.1, 10000 g of cells were collected, centrifuged at 4℃for 20 min, and the supernatant was discarded, followed by addition of 25 ml of S30 buffer (10 mM Tris (OAc), 1.4 mM Mg (OAc) ₂ The cells were rinsed 2 times with 60 mM K (OAc) and 2 mM DTT, and the cells were collected and stored at-80℃for use. Weighing 0.6-0.8 g, taking S30 buffer pre-cooled by 1 ml and 0.1 mL glycerol to resuspend cells, performing ultrasonic crushing (phi 6 ultrasonic probe, 50% power, ultrasonic 2S, stop 6S, total time 6-8 min), and centrifuging at 10000 rpm and 4 ℃ for 30 min. The supernatant was dispensed into 1.5 mL EP tubes and stored at-80℃until use.

(2) After optimization, rhodococcus CFPS reaction system and reaction conditions: 1.2 mM ATP; GTP, UTP and CTP each 0.85 mM; 34. mu g/mL folinic acid; 12-18 ng/. Mu.L plasmid, 0.6-0.8U/uLT RNA polymerase, 0.1-0.2U/uL RNase inhibitor; 20 standard amino acids each 3 mM, 0.33 mM Nicotinamide Adenine Dinucleotide (NAD); 0.27 mM coenzyme A (CoA); 1.5 mM spermidine; 1 mM putrescine; sodium 4 mM oxalate; 290 mM potassium glutamate; 10 mM ammonium glutamate; 6 mM magnesium glutamate; 57 mM HEPES; 1-2% PEG8000pH 7.2;67 mM phosphoenolpyruvate (PEP) and 25-33% (v/v) of a cell extract, and incubating at 15-20℃for about 20 h.

According to the rhodococcus CFPS system, eGFP is expressed and the expression quantity is detected, the experimental result is shown in figure 3, the optimized reaction system has very strong fluorescence brightness, the system almost has no green fluorescence before optimization, and the system shows obvious green fluorescence after optimization, which indicates that the expression quantity of eGFP is obviously improved. The enzyme-labeled instrument detects the fluorescence value and calculates the eGFP concentration, and the comparison between the fluorescence value and the eGFP concentration before and after the system optimization is as shown in FIG. 4, under the optimal condition, the eGFP expression quantity of the CFPS is 322 ug/mL, which is improved by about 10 times compared with the initial value, and the eGFP expression quantity of the CFPS is above 300 ug/mL through the verification of three repeated experiments, which indicates that the method is repeatable.

Claims (8)

1. A system for cell-free protein synthesis in rhodococcus comprising the following components:

(1) PEG8000 addition: 1-2% w/v;

(2) Addition amount of rhodococcus cell extract: 25-33% v/v;

(3) The addition amount of the DNA template: 12-18 ng/uL plasmid;

(4) The addition amount of the RNase inhibitor: 0.1 to 0.2U/uL;

(5) Addition amount of T7RNA polymerase: 0.6-0.8U/uL;

and ATP, GTP, UTP and CTP, folinic acid, 20 standard amino acids, nicotinamide adenine dinucleotide; coenzyme A; spermidine; putrescine; sodium oxalate; potassium glutamate; ammonium glutamate; magnesium glutamate; HEPES; phosphoenolpyruvic acid;

the rhodococcus cell extract is prepared by the following method:

(1) And (3) culturing the rhodococcus, namely fermenting and culturing the rhodococcus by adopting a fermentation medium, wherein the fermentation medium comprises the following components: 10-50 g of glucose, 1-10 g of yeast powder, 1-8 g of peptone, 1-8 g of malt extract, 0.1-1 g of dipotassium hydrogen phosphate, 0.1-1 g of monopotassium phosphate, 0.5-3 g of magnesium sulfate, 0.5-3 g of urea and water to a constant volume of 1L, wherein the pH is 7.2, and the temperature is 115 ℃ for 20 min;

(2) And (3) thallus collection: when fermenting and culturing to OD of thallus ₆₀₀ Collecting thalli in 1.9-3.1; and adjusting the concentration of the bacterial cells to be 0.6-0.8 g/mL;

(3) And (3) thallus crushing: the method comprises the steps of adopting ultrasonic bacteria breaking, wherein the ultrasonic conditions are that the power is 50%, the ultrasonic is 2 s, the interval is 6 s, and the total time is 6-8 min, so as to obtain a cell extract; wherein glycerol is added before sonication or dithiothreitol is added after sonication.

2. The system for cell-free protein synthesis according to claim 1, wherein in the step (3), glycerol added before sonication means 10% glycerol added before sonication.

3. The system of claim 1, wherein the following components are added in a final concentration: ATP 1.2 mM; GTP, UTP and CTP are each 0.80-0.90 mM;30-40 μg/mL folinic acid; 2.5-3.5 mM of 20 standard amino acids each; 0.30-0.36 mM nicotinamide adenine dinucleotide; 0.25-0.30 mM; 1.0-2.0. 2.0 mM spermidine; 0.5-1.5 mM putrescine; 2-6 mM sodium oxalate; 280-300 mM potassium glutamate; 8-12 mM ammonium glutamate; 4-8 mM magnesium glutamate; 50-60 mM HEPES pH 7.2; 65-70 mM phosphoenolpyruvic acid.

4. The system of claim 1, wherein the ingredients are added in a final concentration of: ATP 1.2 mM; GTP, UTP and CTP each 0.85 mM;34 μg/mL folinic acid; PEG 8000% w/v;12 ng/. Mu.L plasmid; 0.8 U/uL T7RNA polymerase; 0.2 U/uL RNase inhibitors; each 3 mM of the 20 standard amino acids; 0.33 mM nicotinamide adenine dinucleotide; 0.27 mM; coenzyme A;1.5 mM spermidine; 1 mM putrescine; sodium 4 mM oxalate; 290 mM potassium glutamate; 10 mM ammonium glutamate; 6 mM magnesium glutamate; 57 mM HEPES pH 7.2;67 mM phosphoenolpyruvate and 33% Rhodococcus cell extract.

5. The system of any one of claims 1 to 4, wherein the pH of the system is 7.0-7.4.

6. A method for cell-free protein synthesis in rhodococcus, characterized in that the system according to any one of claims 1 to 5 is used for reaction at 15 to 20 ℃ for 20 to 44 hours.

7. Use of the system according to any one of claims 1 to 5 or the method according to claim 6 in cell-free protein synthesis.

8. Use according to claim 7, for rapid protein synthesis, rapid construction of enzyme mutation libraries, genetic circuit prototypes or metabolic pathway construction.

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