WO2022193433A1

WO2022193433A1 - Application of system in improving insertion efficiency of unnatural amino acids

Info

Publication number: WO2022193433A1
Application number: PCT/CN2021/095420
Authority: WO
Inventors: 齐浩
Original assignee: 天津大学
Priority date: 2021-03-15
Filing date: 2021-05-24
Publication date: 2022-09-22
Also published as: CN113322267A; CN113322267B

Abstract

An application of a system in improving insertion efficiency of unnatural amino acids. The system can degrade termination factors (RF1, RF2, ArfB, and Pth) in an Escherichia coli translation system and generates a codon-independent translation termination mechanism. The system can degrade the termination factors in the Escherichia coli translation system and can improve insertion efficiency of simultaneously inserting various unnatural amino acids into a target protein. On a protein level, the termination factors in the Escherichia coli translation system can be completely degraded. After the termination factors in the Escherichia coli translation system are degraded, unnatural amino acids can be inserted into TAG, TGA and TAA codons, thereby realizing insertion of the unnatural amino acids into the three termination codons.

Description

Application of the system in improving the insertion efficiency of unnatural amino acids

This application claims the priority of the Chinese patent application with the application number of 202110276679.7 and the title of the invention "Application of the System in Improving the Insertion Efficiency of Unnatural Amino Acids", which was submitted to the China Patent Office on March 15, 2021, the entire contents of which are by reference Incorporated in this application.

technical field

The invention relates to the field of genetic engineering, in particular to the application of the system in improving the insertion efficiency of unnatural amino acids.

Background technique

In nature, 20 kinds of natural amino acids can produce natural proteins with diverse structures and functions, but with the development of biotechnology, the original proteins can no longer meet their needs in protein drug production and enzyme stability. Studies have found that the genetic code of organisms also has certain differences between different species. For example, in the mitochondria of Saccharomyces cerevisiae, the stop codon UGA can also encode tryptophan. In many species, including humans, UGA can also be used to encode unnatural amino acids (UNAAs) other than conventional amino acids, such as selenocysteine, phosphoserine, acetylphenylalanine Wait. These UNAAs can endow proteins with new chemical properties, structures and functions, opening the door to new protein engineering and providing new avenues for biological research, biotherapeutics and synthetic biology.

Some researchers have used codon expansion technology to insert unnatural amino acids into antibodies. The core is to introduce a set of orthogonal exogenous translation tools, that is, aminoacyl-tRNA synthetase that can specifically recognize unnatural amino acids and can recognize Paired tRNAs with stop codons form an orthogonal pair of aminoacyl tRNA synthetase/tRNA. At the same time, the introduced orthogonal system cannot cross-react with the endogenous aminoacyl-tRNA synthetase in the host cell, nor with the tRNA in the host cell. Axup et al. used para-acetylphenylalanine (pAcF) aminoacyl-tRNA synthetase/tRNA _UAG ^pAcF orthogonal molecular pair to insert para-acetylphenylalanine into the variable region of the antibody to prepare a coupling ratio of about 2. Antibody drug conjugates. Ashok et al. used the aaRS/tRNA orthogonal system to insert dopa into the active center of alcohol dehydrogenase II to improve the antioxidant capacity of the enzyme. Unnatural proteins are primarily synthesized intracellularly at first, and cells not only need to maintain a growth state to synthesize target proteins with the highest yield, but due to the complex intracellular metabolic pathways, the intracellular synthesis of proteins containing unnatural amino acids usually leads to product yields. At the same time, most of the unnatural amino acids are toxic to cells to varying degrees and are not easy to pass through the cell membrane, which reduces the content of the target protein. These limitations limit the wide application of intracellular synthesis of unnatural proteins. At the same time, the cell-free protein synthesis (CFPS) system is becoming more and more mature, which promotes the development of extracellular synthesis of unnatural proteins. The cell-free unnatural protein synthesis system is a system for synthesizing unnatural proteins in vitro by adding substrates, energy, cofactors, orthogonal systems, NCAAs and enzymes to cell extracts as templates with exogenous genes. Compared with the intracellular protein synthesis system, the system has the advantages of controllability, short cycle and high system stability.

At present, the main method for inserting NCAAs into CFPS is stop codon suppression based on the natural translation system, which essentially uses stop codons to encode amino acids. However, the introduction of NCAAs into CFPS is limited by the endogenous release factor (RF). ), which mainly recognizes stop codons and causes the release of peptide chains and ribosomes from mRNA, thereby inhibiting the insertion of NCAAs. In cells RF1 is required for translation termination of more than 300 genes in the genome, and direct deletion of the prfA gene can severely affect cell growth and even lead to cell death.

At present, there are various methods to improve the insertion efficiency of unnatural amino acids (UNAAs) in bacteria. For example, after bacterial lysis, endogenous amino acids and endogenous tRNAs are removed by dialysis, and then unnatural amino acids or corresponding This method increases the insertion efficiency of unnatural amino acids to a certain extent. Since the release factor RF is not removed, it will still cause premature termination of translation to a certain extent, resulting in a truncated protein. By engineering chassis cells, all the codons UGA on the E. coli genome were replaced by UAA, and RF1 was knocked out to improve the insertion efficiency of unnatural amino acids during protein translation. Although this method removes the release factor RF1 at the source , but the terminator of translation in bacteria is not limited to RF1.

The researchers used affinity tag, RF1 antibody and mf-lon protease to degrade RF1 protein containing mf-ssrA tag to remove RF1 from cell-free protein synthesis (CFPS). It degrades RF1 horizontally, thereby reducing its inhibition of amber codons and improving the insertion efficiency of unnatural amino acids. However, due to the protein translation process, the termination of protein translation is not only the effect of RF1, so there are still some competing factors. Inhibits the insertion of NCAAs in CFPS; and with current methods, simultaneous insertion of multiple NCAAs is less efficient.

Therefore, it is of great practical significance to provide a method for preparing multiple unnatural amino acid proteins based on genetic modification.

SUMMARY OF THE INVENTION

In view of this, the present invention provides the application of the system in improving the insertion efficiency of unnatural amino acids. The invention realizes the simultaneous insertion of multiple and multiple unnatural amino acids into the protein, wherein the insertion efficiency of 2 unnatural amino acids is 17%, the insertion efficiency of 3 unnatural amino acids is more than 24%, and the insertion efficiency of 12 unnatural amino acids is more than 24%. Amino acids, 69% efficient,

It can endow the protein with more properties and expand the application scope of the protein.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides the application of mf-lon protease mediated by mf-ssrA tag in directional degradation of termination factors in protein translation.

In some specific embodiments of the invention, the termination factor comprises one or more of RF1, RF2, ArfB or Pth.

In some specific embodiments of the invention, the protein is translated into the E. coli protein translation system.

The present invention also provides the use of mf-lon protease mediated by the mf-ssrA tag in improving the insertion efficiency of multiple and/or multiple unnatural amino acids.

In some specific embodiments of the invention, the unnatural amino acid comprises one or more of Bock, pAzF or Sep.

In some specific embodiments of the invention, the plurality includes 2, 3 or 12.

The present invention also provides the application of mf-lon protease, mediated by mf-ssrA tag, in degrading the termination factor in Escherichia coli translation system and improving the efficiency of inserting multiple and/or multiple unnatural amino acids into the target protein ; The termination factor includes one or more of RF1, RF2, ArfB or Pth; the unnatural amino acid includes one or more of Bock, pAzF or Sep; the multiple includes 2, 3 or 12.

Based on the above, the present invention also provides a system for improving the insertion efficiency of multiple and/or multiple unnatural amino acids, including mf-lon protease and mf-ssrA tag.

In some specific embodiments of the invention, the system further comprises an elastin-like polypeptide (ELP).

The present invention also provides the application of the system in the directional degradation of protein translation termination factors; the termination factors include one or more of RF1, RF2, ArfB or Pth.

The present invention also provides the application of the system in improving the insertion efficiency of multiple and/or multiple unnatural amino acids; the unnatural amino acids include one or more of Bock, pAzF or Sep; the multiple includes 2, 3 or 12.

The beneficial effects of the present invention include but are not limited to:

1. The termination factors (RF1, RF2, ArfB and Pth) in the E. coli translation system can be degraded, resulting in a codon-independent translation termination mechanism.

2. Degrade all termination factors in the E. coli translation system, which can improve the insertion efficiency of simultaneously inserting multiple and multiple unnatural amino acids into the target protein;

3. At the protein level, all termination factors in the E. coli translation system can be degraded;

4. After the termination factors in the E. coli translation system are all degraded, unnatural amino acids can be inserted into the TAG, TGA and TAA codons to achieve the insertion of three termination codons of unnatural amino acids.

In some embodiments of the present invention, 12 unnatural amino acids (Bock) can be inserted into the same protein with the help of elastin-like polypeptide (ELP), and the insertion efficiency is as high as 69%, which greatly improves unnatural amino acids. Insertion efficiency of amino acids. In the study of the present invention, elastin-like polypeptide is not necessary, but is required in some embodiments, because the present invention inserts 12 unnatural amino acids in the same protein, which can theoretically be realized in other proteins as well. Since there are many unnatural amino acids inserted in the present invention, suitable sites need to be screened; at the same time, the side chain structure of the unnatural amino acid may affect the spatial structure of the target protein, thereby affecting its function, and the elastin-like polypeptide selected by the present invention is composed of It consists of 15 amino acids. According to the properties of the elastin-like polypeptide, the 14th amino acid can be replaced by any amino acid without affecting its function, so the present invention selects the elastin-like polypeptide.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the schematic diagram of the experiment;

Fig. 2 shows the preparation flow chart of linearization template;

Figure 3 shows Western Blot protein quantification;

Figure 4 shows the release factor RF1 and RF2 test;

Figure 5 shows the detection of insertion activity of unnatural amino acids;

Figure 6 shows the detection of double-plug and triple-plug activity;

Fig. 7 shows the detection of dodecadal activity;

Figure 8 shows the effect of termination factors.

Detailed ways

The invention discloses the application of the system in improving the insertion efficiency of non-natural amino acids, and those skilled in the art can learn from the content of this paper and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The Lon protease (mf-Lon) of Mycoplasma can specifically recognize the mf-ssrA tag (pdt3 tag) without being degraded by the Lon protease in E. coli. The λ-Red-mediated recombination technology system, using the E. coli exonuclease and I-SceI homing endonuclease in the pTKRED plasmid, can insert genes into any position of the E. coli chromosome. The present invention integrates the Lon protease degradation system and the λ-Red-mediated recombination technology system, and inserts the mf-ssrA gene after the target gene. The plasmids expressing mf-Lon and o-aaRS/o-tRNA were transformed into host bacteria, and the bacteria were cultured under the induction of arabinose. After the bacteria were disrupted, S30 extract was prepared, and the insertion efficiency of unnatural amino acids was determined.

The invention utilizes mf-lon protease under the mediation of mf-ssrA tag (namely, the pdt#3 tag research group has applied for patent CN201910763138.X), to directionally degrade key factors in the translation process of several proteins, and improve various unnatural amino acids. insertion efficiency.

The release factors RF1 and RF2 were degraded using the above system to improve the insertion efficiency of unnatural amino acids.

The above system was used to degrade the termination factors (RF1, RF2, ArfB and Pth) in the E. coli translation system, resulting in a codon-independent translation termination mechanism.

Using the above system, essential gene products of E. coli can be degraded at the protein level to improve the insertion efficiency of unnatural amino acids.

Using the above system, any protein of interest in E. coli can be knocked out at the protein level.

Using the above system, the termination factor in the E. coli translation system was degraded to realize the insertion of three unnatural amino acids with termination codons.

Using the above system, the termination factor in the E. coli translation system is degraded, and the efficiency of inserting various unnatural amino acids into the target protein is improved.

The system is not limited to E. coli, but can also be applied to other microbial cell-free technologies.

In the application of the system provided by the present invention in improving the insertion efficiency of the unnatural amino acid, the raw materials and reagents used can be purchased from the market.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 Using λ-Red system to insert pdt3 tags on RF1 and RF2 genes

1. Preparation of electrotransformation competence:

The cryopreserved BL21 Star (DE3) strain was diluted with LB medium at a ratio of 1:100,000, coated with a spreader, and then placed in a 37°C constant temperature incubator for overnight culture, and a single colony was picked and placed in an antibody-free place. After 12 hours, it was transferred to 3 ml of LB medium without antibody. When the OD600 reached 0.6, it was placed on ice for 15 minutes, centrifuged at 4000 rpm/min at 4°C for 3 minutes, and the supernatant was discarded. Then, use 1 ml of sterilized water to reselect the bacterial cells, centrifuge at 4000rpm/min for 3 min at 4°C, repeat once, and then resuspend with 100ul of 10% glycerol, quick-freeze in liquid nitrogen, and store at -80°C.

2. Preparation of Linearization Template

Using the genome of the BL21 Star (DE3) strain and the pTKS/CS plasmid as templates, PCR reactions were carried out according to the following steps: a total of 4 rounds of PCR reactions were required:

The first round of PCR reaction:

reaction system:

Table 1

Table 2

Reaction conditions:

table 3

Second round PCR reaction:

reaction system:

Table 4

Reaction conditions:

table 5

The third round of PCR reaction:

Reaction conditions:

Table 6

Reaction conditions:

Table 7

Fourth round PCR reaction:

Reaction conditions:

Table 8

Reaction conditions:

Table 9

At the end of each step of PCR reaction, perform agarose gel, and then use cutting gel to recover the corresponding target fragments. First, weigh a clean 1.5ml centrifuge tube, then cut the target fragments under blue light, and place them in the corresponding centrifuge tubes. After weighing, add 100ul of sol solution according to 0.1g and place it in a metal bath at 56°C. After the gel is completely melted, put it on ice for 2min, transfer it to a preparation tube, 12000rpm/min for 1min, discard the liquid, and repeat this process. Step until all the liquid is transferred to the preparation tube, then add 600ulbufferW, 12000rpm/min for 1min, discard the liquid, repeat once and then empty centrifuge for 2min; then add 50ulddH2O to elute. After the concentration was measured, it was stored at -20°C.

3. Transform the pTKRED plasmid:

Take out the electrotransformation competent BL21 Star (DE3) and thaw it on ice, add 5ulpTKRED plasmid, put it on ice for 10min, use an electroporator to transfer the large fragment into the strain, then add 1ml SOB medium, and activate it at 30°C for 1h. Spread on LB plates of 100ug/ml Spe, and culture at 30°C in the dark. Visible colonies were seen after 20h. A single colony was picked and cultured in LB medium containing 100ug/ml Spe. When the OD600 reached 0.6, the electrotransformation competent BL21Star(DE3)-RED plasmid was prepared according to the above steps.

4. Convert large fragments:

Take out the electrotransformation competent BL21 Star(DE3)-RED plasmid and melt it on ice, add 5ul of the purified large fragment above, put it on ice for 10 min, use the electroporator to transfer the large fragment into the strain, and then add 1 ml of SOB to cultivate After activation at 30 °C for 1 h, it was spread on LB plates with 2 mM IPTG, 100 ug/ml Spe and 20 ug/ml Tet, and cultured at 30 °C in the dark. Visible colonies were seen after 20h. Pick a single colony and cultivate it in LB medium containing 2mM IPTG, 100ug/ml Spe and 20ug/ml Tet, and after culturing for 1h, carry out bacterial inspection:

Reaction conditions:

Table 10

Reaction steps:

Table 11

After the PCR reaction was completed, agarose gel electrophoresis was performed for verification.

5. Removal of Tet resistance:

Pick the correct bacterial strains and inoculate them in 3ml LB (2mM IPTG, 0.2% arabinose and 100ug/ml Spe), and cultivate at 30°C for more than 15h. The bacterial solution was streaked on LB plates of 2mM IPTG, 0.2% arabinose and 100ug/ml Spe. After culturing at 30℃ for about 24h, obvious colonies were obtained. Pick colonies for PCR verification: the reaction steps are the same as those in 4. The PCR product of F3-LR was sent for sequencing.

6. Removal of pTKRed plasmid:

The correctly sequenced strains were drawn on LB plates with Spe 100ug/ml and cultured at 30°C. Pick a single colony in 3 ml of LB without antibody, culture at 42 °C for 5 h, take the bacterial solution and streak it on an antibody-free plate, and culture at 37 °C overnight. Three single colonies were selected and cultured overnight in LB without anti-LB and Spe100ug/ml respectively. If the Spe is not long, it proves that it has been removed and the bacteria are preserved.

7. Other factors causing translation termination (ArfB and Pth) were also tagged using the above steps, and the factors causing translation termination (ssrA and ArfA) were knocked out using the above steps.

8. Finally, according to the above steps, the SfB-ABCFPTfA strain without translation termination factors (RF1, RF2, ssrA, ArfA, ArfB and Pth) was constructed

Table 12 Primer sequence list

The preparation of embodiment 2 S30 extract and the inspection of RF1 and RF2 in bacteria

1. Preparation of electrotransformation competence and bacterial culture:

The correctly sequenced BL21 Star(DE3)-SLA3B3 strain was prepared according to the above steps to prepare the BL21 Star(DE3)-SLA3B3 electrotransformation competent, and then the pZA16mflon-pAzFRS plasmid was electroporated into BL21 Star(DE3)-SLA3B3, and then plated (Amp- LB plate), cultivated overnight at 37 °C; single colonies were picked from the plate, inoculated into 22 mL of LB liquid medium, and incubated overnight at 37 °C on a shaker for 10 hours. To take a part of the bacteria need to be cryopreserved. Take 10 mL of overnight bacterial solution, inoculate it in 1.0 L of S30 medium containing 1 mM arabinose and 1 mL of 100 mg/ml ampicillin sodium, and culture at 37°C for about 11 h until OD ₆₀₀ =1.7 or so to harvest bacteria.

2. Collection of bacteria

① Immediately after the incubation, place the bacterial solution in an ice bath, stand still for 30 minutes, weigh the empty 50ml centrifuge tube, and mark it;

②The cells were collected by centrifugation (4200rpm, 4°C, 30min).

③ Add 30mL S30 buffer to resuspend and wash the bacteria, and transfer to a 50mL centrifuge tube, then collect the bacteria by centrifugation (5000g, 4°C, 15min). This step needs to be repeated once;

④ Fully discard the supernatant, weigh the centrifuge tube, and calculate the weight of the wet cells (total weight - net centrifuge tube weight);

⑤Put the cells into -80°C freezer and store.

S30 buffer formula:

Table 13

3. Preparation of S30 extract:

①Take out the preserved cells from -80°C and thaw them in an ice bath. After thawing, add S30 buffer according to the ratio of 1.1 g (110%) wet cells to 1 mL of S30 buffer, and resuspend the cells completely.

②Pre-cool the instrument, adjust the pressure to 1655bar (24000psi), crush once,

③ Collect the supernatant by centrifugation (30000g, 4℃, 30min)

④ Repeat the above steps once

⑤run-off: transfer the supernatant to a 15ml centrifuge tube, wrap it in tin foil, and incubate at 37°C at 220rpm/min for 1h;

⑥ Collect the supernatant by centrifugation (30000g, 4℃, 30min)

⑦ The collected supernatant, obtained BL21Star(DE3)-SLA3B3 extract, was subpackaged, snap-frozen in liquid nitrogen, and stored at -80°C.

4. Inspection of RF1 and RF2

Sample processing: Take 1ml of the above cultured cells, centrifuge the cells at 8000rpm/min for 2min to collect the cells, wash the cells once with S30 buffer, then add 60ul S30 buffer and 12ul 6×SDS Loading Buffer and mix well, then place in a 98°C metal bath Boil for 10min in medium and centrifuge at 12000r/min at 4°C for 1min. Take 15μL of the above-mentioned two centrifugation broken liquid (before run-off) and the supernatant collected after the last run-off, add 3μL of 6×SDS Loading Buffer, mix well, put it in a metal bath at 98°C and boil for 10min, 12000r/min4 Centrifuge for 1 min.

After loading, after SDS-PAGE gel electrophoresis, transfer to PVDF membrane by wet transfer (100V-120min), add 4mL of 5% BSA to block for 2h, wash 4 times with TBST, 10min/time, add diluted The primary antibody (rabbit anti-Flag-labeled monoclonal antibody) was incubated for 2h, washed 4 times with TBST, 10 min/time, and then added with the diluted secondary antibody (HRP-labeled goat anti-rabbit polyclonal antibody), incubated for 2h, washed 4 times with TBST, 10min/time, remove the membrane and add 0.8mL of ECLplus luminescent solution, incubate for 1min, and develop.

Western Blot results showed that when the protein loading amount was the same (Figure 3), compared with the uninduced group, the intracellular contents of release factors RF1 and RF2 in the whole cell were significantly reduced, indicating that the LR system was successfully constructed; Following bacterial disruption, the synthesis of release factors RF1 and RF2 was blocked at 30,000 g and 4°C, but the induced mf-lon protein could still be active, and release factors RF1 and RF2 were continuously degraded. Finally, after two centrifugations, The release factors RF1 and RF2 were completely degraded (Figure 3).

Example 3 Determination of the activity of S30 extract

1. Preparation of GFP-WT, GFP-149TAG, GFP-149TGA and GFP-149TAA templates

Take the pET23a-GFP, pET23a-GFP149TAG, pET23a-GFP149TGA and pET23a-GFP149TAA strains stored in the laboratory, streak them on the plate, and then put them into 37 ℃ culture for 14h, pick a single colony, put it in 5ml of LB (100ug /mlAmp), 220rpm/min at 37°C for 14h, and then extract the plasmid according to the kit instructions. The plasmid was subjected to PCR reaction according to the following steps:

Table 14

Reaction conditions:

Table 15

After the PCR reaction was completed, agarose gel electrophoresis was performed, and the gel was cut and recovered according to the steps described above. After the concentration was measured, it was stored at -20°C.

2. Detection of the activity of S30 extract

①Preparation of 20×Amino Acid (20×AA): According to the following table, configure 20×Amino Acid (20×AA), please add it from top to bottom in the order in the table.

Table 16

②Configuration of 20×PEP

Table 17

③25×Nucleotide Mix(25×NM)

Table 18

④ Prepare 10×Salt Mix (10×SM)

Table 19

⑤ Prepare 4×Buffer Mix: configure 4×Buffer Mix according to the following table

Table 20

⑥ Mix T7RNAP (preserved in the laboratory) and 4×BufferMix according to the following table, then add the corresponding reagents in order from top to bottom, and finally add the natural amino acid. In the standard instrument, the reaction temperature was set to 30 °C, the reaction was performed for 3 h, and the GFP expression was detected with 485 nm as the excitation wavelength and 535 nm as the emission wavelength.

Table 21

Determination of the activity of S30 extract:

The fluorescence results showed that the cell-free protein expression system prepared by using the S30 extract could insert the unnatural amino acids Bock, pAzF and Sep into the GFP protein, indicating that the CFPS system was successfully constructed; compared with the control group, the knockout release factor RF1 and RF2, can significantly improve the insertion efficiency of unnatural amino acids; by examining the insertion of different stop codons, it can be seen that after knocking out the release factors RF1 and RF2, the three unnatural amino acids Bock, pAzF and Sep can be inserted into different The stop codons of 3 stop codons were inserted into unnatural amino acids (Fig. 5, Table 22).

Table 22

3. Detection of double-plug and triple-plug activity

According to the above-mentioned method for preparing the extract, the extract was prepared with the SfB-ABCFPTfA strain as the host bacteria. Mix T7RNAP (experimental preservation) and 4×BufferMix according to the above table, then add the corresponding reagents in order from top to bottom, and finally mix two NCAAs (Bock and pAzF) or three (Bock, pAzF and Sep) Add to the system, mix well and add 18ul to the 384 plate, put it into the microplate reader, set the reaction temperature to 30°C, and react for 3h, with 485nm as the excitation wavelength and 535nm as the emission wavelength to detect the expression of GFP.

The fluorescence results showed that compared with the control group, knocking out the release factors RF1 and RF2 could significantly improve the insertion efficiency of double insertion (Bock and pAzF) and triple insertion (Bock, pAzF and Sep) in CFPS, and the insertion efficiency was as high as 50%. above (Fig. 6, Table 23).

Table 23

4. Detection of Dodecanal Activity

① Template preparation:

Take the pET23a-ELP, pET23a-ELP12TAG-GFP, pET23a-ELP6(TAG TGA)-GFP, pET23a-ELP4(TAG TGA TAA)-GFP and pET23a-ELP triple insert-GFP mutant strains preserved in the laboratory, and carry out plate streaking , then placed in 37°C culture for 14h, single colonies were picked, placed in 5ml of LB (100ug/ml Amp), 220rpm/min at 37°C for 14h, and then the plasmids were extracted according to the kit instructions. The plasmid was subjected to PCR reaction according to the following steps:

Table 24

Reaction conditions:

Table 25

②According to the above-mentioned method for preparing the extract, the extract was prepared with the SfB-ABCFPTfA strain as the host bacteria. Mix T7RNAP (preserved in the laboratory) and 4×BufferMix according to the above table, then add the corresponding reagents from top to bottom in order, and finally add the three unnatural amino acids (Bock, pAzF and Sep) into the system, mix well Then, 18ul was added to the 384 plate, placed in a microplate reader, the reaction temperature was set to 30°C, and the reaction was performed for 3h. The expression of GFP was detected with 485nm as the excitation wavelength and 535nm as the emission wavelength.

The fluorescence results showed that compared with the control group, knocking out the release factors RF1 and RF2 could significantly improve the insertion efficiency of Twelve in CFPS (Figure 7, Table 26).

Table 26

5. Expression of target gene without terminator

Take the pET23a-GFP strain stored in the laboratory, streak the plate, and then put it into 37°C culture for 14h, pick a single colony, put it in 5ml of LB (100ug/ml Amp), and cultivate at 220rpm/min at 37°C for 14h , and then extract the plasmid according to the kit instructions. The plasmid was subjected to PCR reaction according to the following steps:

Table 27

Reaction conditions:

Table 28

According to the above method for preparing the extract, the SfB-ABCFPTfA strain was used as the host bacteria to prepare the extract, and T7RNAP (preserved in the laboratory) and 4×BufferMix were mixed together according to the above table, and then backfilled with RF1, RF2, ArfB and Pth respectively Stop factor, and finally add the corresponding reagents from top to bottom according to the CFPS system, add to the system, mix evenly, add 18ul to the 384 plate, put it into the microplate reader, set the reaction temperature to 30 ℃, and react for 3h, with 485nm as the Excitation wavelength, 535nm is the emission wavelength to detect the expression of GFP.

Fluorescence results showed that RF1, RF2, ArfB, and Pth termination factors were backfilled into the CFPS system to promote the release of ribosomes in the CFPS system, allowing ribosomes to participate in the next round of translation, realizing a codon-independent translation termination mechanism. Among them, backfilling ArfB and Pth terminators into the system can increase the fluorescence value of GFP by more than 2 times (Fig. 8, Table 29).

Table 29

The application of the system provided by the present invention in improving the insertion efficiency of unnatural amino acids has been described in detail above. The principles and implementations of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

Use of mf-lon protease mediated by the mf-ssrA tag for directional degradation of terminators in protein translation.
The application of claim 1, wherein the termination factor comprises one or more of RF1, RF2, ArfB or Pth.
The application of claim 2, wherein the protein is translated into an Escherichia coli protein translation system.
Use of mf-lon protease mediated by mf-ssrA tag in improving the insertion efficiency of multiple and/or multiple unnatural amino acids.
The application of claim 4, wherein the unnatural amino acid comprises one or more of Bock, pAzF or Sep.
6. The use of claim 5, wherein the plurality comprises 2, 3, or 12.
The application of mf-lon protease, mediated by the mf-ssrA tag, in degrading the termination factor in the translation system of Escherichia coli and improving the efficiency of inserting multiple and/or multiple unnatural amino acids into the target protein;

The termination factor includes one or more of RF1, RF2, ArfB or Pth;

The unnatural amino acid includes one or more of Bock, pAzF or Sep;

The plurality includes 2, 3 or 12.
A system for improving the efficiency of insertion of multiple and/or multiple unnatural amino acids, characterized by comprising mf-lon protease and mf-ssrA tag.
Use of the system of claim 8 for directional degradation of termination factors in protein translation; the termination factors include one or more of RF1, RF2, ArfB or Pth.
The application of the system according to claim 8 in improving the insertion efficiency of multiple and/or multiple unnatural amino acids; the unnatural amino acids include one or more of Bock, pAzF or Sep; the multiple Include 2, 3 or 12.