CN116103210B - System for efficient cell-free in-vitro protein expression - Google Patents

Abstract

The invention relates to a method for establishing a cell-free expression system of escherichia coli, which can economically and rapidly realize the high-efficiency expression of fluorescent protein eGFP without adding tRNA, T7 RNA polymerase, a cell crowding agent and various kinase components, can realize the expression level of 3.35 mg/mL in 2 hours, and has important research significance and application value.

Description

System for efficient cell-free in-vitro protein expression

Technical Field

The invention belongs to the technical field of biology, and relates to a system for cell-free in vitro expression of proteins.

Background

Cell-free protein synthesis (cell-free protein synthesis, CFPS) is also known as in vitro protein transcription and translation technology, and this concept was first proposed in the 60 s of the 20 th century and has been developed over 60 years to date. Compared with in vivo expression technology, the technology does not need transfected cells, cell culture and protein purification processes, and only needs plasmids or amplification products of target fragments and raw materials, energy substances, elements and the like required in transcription and translation processes to start expression, so that the technology has quite superior expression efficiency, and can generally obtain expression products within 2 to 12 hours. And in synthetic biology, cell-free expression is an important in vitro expression modality, often used for the synthesis of proteins that are difficult to achieve in vivo expression, for example: membrane proteins, toxic proteins, and the like.

The CFPS system has a variety, and the most common commercial kits on the market at present comprise an escherichia coli expression system, a wheat germ expression system, a PURE system and the like. In cell-free expression, a protein expression level of greater than 1 mg/mL is considered to be a high-efficiency expression system, which is representative thereof, and techniques and methods for synthesis in a cell-free synthesis system using E.coli as a source of cell extracts are well known in the art. The construction of cell-free expression system platforms for E.coli is detailed in 2021 by David Garenned et al, and includes the preparation of E.coli cell extract, the selection of energy system, the concentration of salts and amino acids, etc.

The expression level of the existing escherichia coli cell-free expression system is generally 0.1-1.5 mg/mL, the level of fluorescent protein deGFP expressed by the escherichia coli cell-free system in 2014 Filippo Casschera et al reaches 2.3 mg/mL, the deGFP expressed by the escherichia coli cell-free system in 2011 Jonghyeon Shin et al reaches the expression level of 3 mg/mL, IPTG is added to improve the expression of T7 RNA polymerase when cell extracts are prepared, and 0.2 mg/mL tRNA and dNTP are added to the expression system, so that the aim of improving transcription and translation efficiency is fulfilled, the cost of cell-free expression is greatly improved, and the method is more suitable for production and application of high-added value products.

Therefore, it is important to establish a cell-free expression system which is economically applicable to most protein expression and can realize efficient expression.

Disclosure of Invention

The technology of the invention relates to a cell-free expression system, and further relates to an escherichia coli cell-free expression system, and the aim of the invention is to provide a method for preparing the escherichia coli cell-free expression system with low cost, high yield and economical applicability, comprising the preparation of escherichia coli cell extracts, the combination of energy regeneration systems and the method for in vitro expression.

The invention firstly provides a preparation method for stably obtaining a high-activity escherichia coli cell extract, which comprises the following steps:

s1, culturing escherichia coli by adopting a CFPS culture medium, wherein the preparation method of the CFPS culture medium comprises the following steps: 18-22 g peptone, 4-6 g yeast extract, 4-6 g sodium chloride, 3-5 g lactose, 0.4-0.6 g anhydrous glucose, 4-8 mL glycerin adjusting pH to 7.2-7.4, then fixing volume to 700 mL, sterilizing 8-10 g dipotassium hydrogen phosphate and 2-4 g potassium dihydrogen phosphate to 300 mL, and sterilizing; mixing the two materials under aseptic condition after sterilization;

s2 culturing to OD ₆₀₀ Centrifugally collecting thalli when the temperature reaches 3.0-4.0;

s3, after re-suspending the thalli, ultrasonically crushing the thalli, adding dithiothreitol at least once before using, namely adding the dithiothreitol into the crushing liquid, uniformly mixing, centrifuging to obtain supernatant, namely the cell extract in the reaction system, and directly using or freezing at the temperature of minus 80 ℃ for later use.

Preferably, the preparation method of the CFPS medium comprises the following steps: 20 g peptone, 5 g yeast extract, 5 g sodium chloride, 4 g lactose, 0.5 g anhydrous glucose, 6 mL glycerol, adjusting pH to 7.2-7.4, fixing volume to 700 mL,115 deg.C, and sterilizing for 30 min; 9.13 g dipotassium hydrogen phosphate and 3 g potassium dihydrogen phosphate are sterilized after being fixed to 300 mL and 121 ℃ for 20 min; and after the sterilization, the two components are uniformly mixed on an ultra-clean workbench.

Further preferably, the E.coli is E.coli BL21star (DE 3).

Further preferably, culture to OD ₆₀₀ And centrifugally collecting thalli when the temperature reaches 3.4-3.6.

Further preferably, in S3, the concentration of dithiothreitol is 10-15mM and the centrifugation is performed at 10000-14000 rpm to obtain a supernatant.

To achieve increased levels of protein expression in vitro, conventional approaches have added additional T7 RNA polymerase and tRNA components to increase transcription and translation efficiency, and molecular crowding agents such as PEG8000 to mimic the cellular environment, which are expensive. The invention improves the components of the culture medium: besides the conventional yeast powder, peptone, salts and glycerol, lactose and glucose are added, lactose can replace the action of IPTG, the expression level of T7 RNA polymerase at the cell background is improved, no additional addition is needed, the cell synthesizes the T7 RNA polymerase, and the tRNA content in the cell body is increased, so that the addition of tRNA is omitted, and the cost of in-vitro expression is reduced.

The invention further provides a system for efficiently expressing proteins without cells, which comprises the following components: HEPES, PEP, NAD, oxalic acid, spermidine, NMP mixed solution, ATP, amino acid mixed solution, in vitro expression plasmid carrying target protein gene as template, potassium glutamate, magnesium glutamate, and Escherichia coli cell extract obtained by the preparation method.

Preferably, the system comprises the following components: 140-160 mM HEPES pH7.5, 30-36 mM PEP, 0.3-0.5 mM NAD, 3-5 mM oxalic acid, 1.2-1.8 mM spermidine, NMP mixture with respective NMP concentrations of 4-6 mM, 2.5-2.7 mM ATP, 1.5-2.5 mM amino acid mixture, 3-7 ng/uL template, 28-37% (v/v) E.coli cell extract, 90-110 mM potassium glutamate and 6-10 mM magnesium glutamate.

More preferably, the system comprises the following components: 140 HEPES pH7.5, 33 mM PEP, 0.4 mM NAD, 4 mM oxalic acid, 1.5 mM spermidine, NMP mixture with respective NMP concentrations of 5mM, 2.6 mM ATP, 2 mM amino acid mixture, 5ng/uL template, 33% (v/v) E.coli cell extract, 100 mM potassium glutamate and 8 mM magnesium glutamate.

The invention further provides a method for expressing target protein by using the system, which comprises the following steps: the system is reacted at 400-600 rpm and 28-32 deg.c for 1-3 h to obtain target protein.

Thus, the invention also provides the use of said system for the production of proteins.

The present invention is directed to the conversion of AMP/GMP/CMP+ATP by replacing 4 conventional nucleoside triphosphates ATP/UTP/GTP/CTP with a combination of nucleoside monophosphates AMP/UMP/GMP/CMP+ATP, wherein AMP/GMP/UMP can be phosphorylated by ATP to their triphosphate form ATP/GTP/UTP, and UTP can be converted to CTP by deamination. ATP is economically readily available relative to the other 3 nucleoside triphosphates, and the price of the other 3 nucleoside monophosphates is much lower than that of the triphosphate form, by a factor of about 30, and the combination AMP/UMP/GMP/cmp+atp greatly reduces costs while ensuring transcription and translation efficiency. In particular, the energy system in an in vitro expression system is necessary to provide four ribonucleosides and regenerate ATP and GTP. Conventional in vitro expression will add ATP and GTP at a concentration of about 1.2-1.5 mM, and CTP and UTP at a concentration of about 0.8-0.9 mM. Without the ATP regeneration system, the yield of in vitro expression would be less than 0.1 mg/mL, with the duration of the expression process also being less than 1 hour. The ATP regenerating system consists of phosphate donors and phosphokinase, the three most commonly used phosphate donors being phosphoenolpyruvate (PEP), phosphoglycerate (3 PGA) and phosphocreatine (CP). Wherein PEP and 3PGA do not require the addition of kinase, they are present in E.coli lysates, and Creatine Kinase (CK) is required to be added to an in vitro expression system together with CP. Thus, in embodiment 3 of the present invention, a relatively low priced PEP was chosen as the phosphate donor, and nucleoside monophosphates were used instead of nucleoside triphosphates, using a combination of nucleoside monophosphates AMP/UMP/GMP/cmp+atp, where AMP/GMP/UMP can be phosphorylated by ATP to their triphosphate form ATP/GTP/UTP, which can be converted to CTP by deamination. ATP is economically readily available relative to other 3 nucleoside triphosphates, nucleoside monophosphates are much less expensive than the triphosphate forms, about 30-fold different, and the combination of AMP/UMP/GMP/CMP+ATP greatly reduces costs while ensuring transcription and translation efficiency.

Drawings

FIG. 1, eGFP protein concentration-fluorescence value standard curve.

FIG. 2 shows the expression of eGFP by SDS-PAGE.

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: construction of eGFP expression vectors

The initial plasmid was pET-28a (+) -eGFP (supplied by Kirsrui Biotechnology Co., ltd.) and was used as a template for inverse PCR using the rapid high-fidelity DNA polymerase PrimeSTAR Max R045A (Takara) to obtain a linear fragment, and after the linear fragment was obtained, the T4 polynucleotide kinase (T4 PNK, NEB) and Ligation high ver.2 (TOYOBO) were used for simultaneous terminal phosphorylation and blunt end Ligation, and finally E.coli was transformed to obtain a circular plasmid with Lac operator deleted, and a plasmid with LacO deleted correctly was obtained by sequencing and named pET-28a (+) -DeltaLacO-eGFP.

25. The composition of the μL PCR amplification system was: 12.5 mu.L PrimeSTAR Max, template 1. Mu.L, 0.75. Mu.L each of the upstream and downstream primers, ddH ₂ O10. Mu.L. The PCR reaction procedure was: pre-denaturation at 98℃for 2 min; denaturation at 98℃for 10 s, annealing at 58℃for 15 s, elongation at 72℃for 90 s,30 cycles; final extension at 72℃for 5 min; preserving at 12 ℃. The system for one-step terminal phosphorylation and blunt-end ligation of PCR products was as follows: ddH ₂ O4. Mu.L, PCR product 5. Mu.L, ligation high 5. Mu.L, T4 PNK 1. Mu.L.

Example 2: routine CFPS expression level verification of escherichia coli

1. Preparation of cell extracts: activating strain BL21Star (DE 3) on solid LB culture medium overnight, picking up monoclonal to 5 mL LB test tube, culturing for 8 hours, transferring to 100 mL 2 XYPT culture medium with 1% inoculum size (16 g peptone, 10 g yeast extract and 5 g sodium chloride to volume of 700 mL,121 ℃ for 20 min for sterilization; 9.13 g dipotassium phosphate and 3 g potassium dihydrogen phosphate to volume of 300 mL,121 ℃ for 20 min for sterilization, mixing the two after cooling, and mixing in an ultra clean bench after sterilization); overnight culture, the next day was transferred to a baffle shake flask of 900 mL 2 XYPT medium at 10% inoculum size, and IPTG was added at a final concentration of 1mM, and the culture was performed at 180 rpm for about 2-3 h with OD ₆₀₀ When the cell count reached 3.0, the cells were collected by centrifugation and resuspended in buffer (10 mM Tris (OAc) pH 8.2, 14 mM Mg (OAc) ₂ 60 mM K (OAc), 2 mM Dithiothreitol (DTT) was added at least once before use.

Then adding bacterial mud with a certain mass into the resuspension buffer, calculating the adding amount according to 0.6 g/mL, calculating an input energy estimation value of ultrasonic crushing according to the volume of bacterial liquid after the bacterial mud is subjected to uniform state, wherein the value is obtained by a related BL21Star (DE 3) ultrasonic crushing energy calculation formula proposed by Yong-Chan Kwon et al in 2015, and setting the input ultrasonic crushing time length by combining the model and the power percentage of an ultrasonic crusher. Such as: adding 5 mL re-suspension buffer into 3 g bacterial sludge, re-suspending, and setting the ultrasonic crushing power to 20% (the instrument model is new sesame JY 88-IIN), and performing ultrasonic treatment for 5s and stopping for 5s for 2 min. And adding 13 mM DTT into the crushed liquid, uniformly mixing, centrifuging at 12000 rpm to obtain a supernatant, namely the cell extract in the reaction system, and subpackaging in a 2 mL centrifuge tube for freezing in a minus eighty-degree refrigerator.

2. E.coli CPFS system configuration and reaction condition setting: the CPFS system contained ingredients including 150 mM HEPES (pH 7.5), 1.5 mM ATP and GTP,0.9 mM CTP and UTP, 0.2. 0.2 mg/mL tRNA,0.4 mM NAD,33 mM PEP,1.5 mM spermidine, 4 mM oxalic acid, 1mM DTT,2% PEG8000,2 mM amino acid mix (see: kwon YC, jewtt MC. High-throughput preparation methods of crude extract for robust cell-free protein synthosis. Sci Rep. 2015 Mar 2;5:8663.), the template (5 ng/uL) obtained in example 1, 33% (v/v) E.coli cell extract, 100 mM potassium glutamate and 8 mM magnesium glutamate. The whole reaction system was reacted at 500 rpm and 30℃for 2h to examine the protein expression. A blank control was set, no plasmid template was added, and other conditions were consistent with the experimental group described above.

3. Protein expression level detection of the escherichia coli CPFS system: the eGFP used in the invention belongs to green fluorescent protein, and can be used for concentration measurement by using a fluorescent quantitative detection method. Firstly, establishing a fluorescence value and protein concentration standard curve, obtaining an eGFP protein sample by adopting an affinity chromatography purification method, determining the protein concentration by utilizing a Bradford protein concentration determination kit, then diluting to a certain concentration gradient (3.74 mg/mL, 2.58 mg/mL, 1.93 mg/mL, 1.6 mg/mL, 1.19 mg/mL, 0.8 mg/mL and 0.44 mg/mL) for fluorescence value detection, respectively setting excitation light and emission light to 485 nm and 528 nm, taking eGFP with different concentrations as an abscissa, and taking an ordinate as a fluorescence value, wherein the measured standard curve is shown in figure 1.

The protein expression level of CPFS system was measured according to the standard method described above, and the sample after 2 hours of reaction was centrifuged at 12000 rpm for 2 minutes, the supernatant was 50. 50 uL, 50 mM HEPES (pH 7.2) was added at 100 uL, the fluorescence value was measured by a microplate reader, and the protein concentration was calculated by a standard curve, and the results are shown in Table 1.

Table 1: eGFP expression in conventional E.coli CFPS

The conventional CFPS expression plasmid is pJL-1, and according to the experimental results in Table 1, eGFP can realize cell-free expression on the modified pET vector, and can be used for an in vitro expression system.

Example 3: influence of the preparation of different cell extracts on the expression level

Conventional high expression CFPS systems would add additional T7 RNA polymerase or IPTG to generate more T7 RNA polymerase to increase transcription efficiency, and tRNA to increase translation efficiency, for the relevant ingredients, see example 2. In fact, the cell itself contains T7 RNA polymerase and tRNA, but the content is not defined. We wanted to explore the effect of cell extracts on protein expression by improving the medium formulation and controlling the level of cell growth. In the experimental process, the cell extracts in different growth stages have larger influence on the protein expression level, and can replace the addition of T7 RNA polymerase and tRNA; the specific experiment is as follows:

first, lactose, glucose and glycerol were added to the 2 XYPT medium components, and the mixture was named CFPS medium. The preparation method comprises the following steps: 20 g peptone, 5 g yeast extract, 5 g sodium chloride, 4 g lactose, 0.5 g anhydrous glucose, 6 mL glycerol, adjusting pH to 7.2-7.4, fixing volume to 700 mL,115 deg.C, and sterilizing for 30 min; 9.13 g dipotassium hydrogen phosphate and 3 g potassium dihydrogen phosphate are sterilized after being fixed to 300 mL and 121 ℃ for 20 min; and after the sterilization, the two components are uniformly mixed on an ultra-clean workbench. The cells were cultured as in example 2, except that IPTG was not added, and the cells were grown at different stages (OD ₆₀₀ 2.0-5.0) are sampled separately.

Cell extracts were prepared by ultrasonication according to the method of example 2, and the reaction system was the same as that of example 2.After expression for 2h, centrifugation was carried out at 12000 rpm for 2 min, and the supernatant was taken at 50. 50 uL, and 50 mM HEPES (pH 7.2) was added at 100. 100 uL, followed by mixing, detection and calculation as in example 2. The results are shown in Table 2, and are divided into a blank control, a positive control and an experimental group, wherein the blank control is not added with a template, and the rest conditions are consistent; positive controls were different OD prepared according to the method of example 2 ₆₀₀ Cell extracts below.

Table 2: different cell extract expression conditions

From the fluorescence values in Table 2, it can be seen that at OD ₆₀₀ The result was the best when eGFP was expressed at 3.5, and the positive control at this time had an expression level of 3.46/mg/mL and the experimental group was 3.23/mg/mL. Accordingly, the method changes the formula of the culture medium and precisely controls the OD of the thalli ₆₀₀ It can realize the self-production of more T7 RNA polymerase and tRNA by cells, thus achieving high-level expression without adding tRNA and T7 RNA polymerase.

Example 4: nucleoside monophosphate energy system

The conventional high expression CFPS system uses nucleoside triphosphates in the form of 1.5 mM ATP and GTP,0.9 mM CTP and UTP, as in example 2, with high cost and poor stability, while nucleoside monophosphates are stable and economical and readily available. It is desirable to achieve the conversion of nucleoside monophosphates to nucleoside triphosphates by adding ATP to the nucleoside monophosphates, and to ensure proper transcription and energy cycling at a lower cost.

The components of the energy system are as follows: 33 mM PEP, 0.4 mM NAD, 4 mM oxalic acid, 1.5 mM spermidine, NMP mixture of NMP at 5mM concentration, 2.0-3.0 mM TP.

In addition to the above energy component addition, the OD of example 3 was added to the reaction system ₆₀₀ The cell extract at 3.5 was added at 33% v/v, with the addition of salts, buffers and amino acid concentrations as in example 2, but without the addition of PEG8000. The reaction system was the same as in step 2 of example 2. Expression 2After h, the mixture was centrifuged at 12000 rpm for 2 min, and the supernatant was taken as 50 uL, and 100 uL of 50 mM HEPES (pH 7.2) was added, followed by mixing, followed by detection and calculation as in step 2) of example 2. The expression results are shown in Table 3 and are divided into a blank control, a positive control and an experimental group, wherein the blank control is not added with a template, and the rest conditions are consistent. The experimental group was set up with 5mM each of NMP and the optimal NMP+ATP combination regimen was explored at different concentrations of ATP.

Table 3: expression of ATP at different concentrations

From the results, it was found that the expression level was not high enough at ATP concentrations of 2.4 or less mM, and that the ATP concentration was low, which could affect the circulation of NMP to NTP. At an ATP concentration of 2.6 mM, the best expression results, corresponding to an expression level of 3.35 mg/mL, are close to 3.56 mg/mL in example 2, indicating that the method is economically viable.

Example 5: SDS-PAGE to verify protein expression

The expression of the experimental group samples obtained in example 4 and the experimental group samples obtained in example 2 was verified by SDS-PAGE, and the samples were diluted 6-fold with 50 mM HEPE (pH 7.5) before treatment, then treated with 2×loading Buffer, heated for 10 min after metal bath at 100deg.C, and loaded with 10 uL. The decolorized protein gel is shown in FIG. 2, wherein eGFP has a size of 27KD, A is the expression of eGFP in the control group, B is the expression of eGFP in the experimental group, and BL21 is the cell extract of BL21star (DE 3).

The eGFP detected by SDS-PAGE shows similar expression level (figure 2), which shows that the expression system in vitro does not need to additionally add tRNA, T7 RNA polymerase and other components, and the high-level expression can be achieved by regulating the components of a culture medium, strictly controlling the OD in the bacterial culturing process and replacing the components of an energy system.

According to the invention, the protein expression quantity can be quantified through a standard curve of fluorescent protein concentration-fluorescent value in the embodiment 2, and after substituting a fluorescent value, the expression level of the pattern protein eGFP can reach 3.56 mg/mL in the conventional method, and can reach 3.35 mg/mL in the embodiment 4, and the difference between the expression level and the expression level is only 0.2 mg/mL, so that the method is efficient and feasible. After SDS-PAGE verification, clear protein bands can be still seen by diluting the eGFP by 12 times, which is far higher than the average level of in-vitro expression of the escherichia coli at present.

Claims (7)

1. A system for efficient cell-free expression of a protein, comprising the following components: the composition comprises the following components: 140-160 mM HEPES pH7.5, 30-36 mM PEP, 0.3-0.5 mM NAD, 3-5 mM oxalic acid, 1.2-1.8 mM spermidine, NMP mixture with respective NMP concentration of 4-6 mM, 2.5-2.7 mM ATP, 1.5-2.5 mM amino acid mixture, 3-7 ng/uL template, 28-37% by volume of E.coli cell extract, 90-110 mM potassium glutamate and 6-10 mM magnesium glutamate;

the preparation method of the escherichia coli cell extract comprises the following steps:

s1, culturing escherichia coli by adopting a CFPS culture medium, wherein the preparation method of the CFPS culture medium comprises the following steps: 18-22 g peptone, 4-6 g yeast extract, 4-6 g sodium chloride, 3-5 g lactose, 0.4-0.6 g anhydrous glucose, 4-8 mL glycerin adjusting pH to 7.2-7.4, then fixing volume to 700 mL, sterilizing 8-10 g dipotassium hydrogen phosphate and 2-4 g potassium dihydrogen phosphate to 300 mL, and sterilizing; mixing the two materials under aseptic condition after sterilization;

s2 culturing to OD ₆₀₀ Centrifugally collecting thalli when reaching 3.4-3.6;

s3, after re-suspending the thalli, ultrasonically crushing the thalli, adding dithiothreitol at least once before using, namely adding the dithiothreitol into the crushing liquid, uniformly mixing, centrifuging to obtain supernatant, namely the cell extract in the reaction system, and directly using or freezing at the temperature of minus 80 ℃ for later use.

2. The system of claim 1, wherein the CFPS medium is prepared by the following method: 20 g peptone, 5 g yeast extract, 5 g sodium chloride, 4 g lactose, 0.5 g anhydrous glucose, 6 mL glycerol, adjusting pH to 7.2-7.4, fixing volume to 700 mL,115 deg.C, and sterilizing for 30 min; 9.13 g dipotassium hydrogen phosphate and 3 g potassium dihydrogen phosphate are sterilized after being fixed to 300 mL and 121 ℃ for 20 min; and after the sterilization, the two components are uniformly mixed on an ultra-clean workbench.

3. The system of claim 1, wherein the E.coli is E.coli BL21star (DE 3).

4. The system of claim 1, wherein in S3, dithiothreitol is at a concentration of 10-15mM and centrifugation is at 10000-14000 rpm to obtain a supernatant.

5. The system according to claim 1, characterized in that it comprises the following components: 140 HEPES pH7.5 mM, 33 mM PEP, 0.4 mM NAD, 4 mM oxalic acid, 1.5 mM spermidine, NMP mixture with NMP concentration of 5mM respectively, 2.6 mM ATP, 2 mM amino acid mixture, 5ng/uL template, 33% by volume of E.coli cell extract, 100 mM potassium glutamate and 8 mM magnesium glutamate.

6. A method for expressing a protein of interest using the system of any one of claims 1-5, comprising the steps of: reacting the system according to any one of claims 1-5 at 400-600 rpm and 28-32 ℃ for 1-3 h, and collecting the target protein obtained by expression.

7. Use of a system according to any one of claims 1-5 for the production of proteins.

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