A method for the synthesis of a cell-free system topoisomerase and its structural monomers

Abstract

The application discloses a method for synthesizing a structure monomer of a topological protein in a cell-free system, comprising the following steps: synthesizing a basic sequence comprising a gene sequence for expressing a target protein, a gene sequence for expressing SpyTag or a gene sequence for expressing SpyCatcher; and adding the basic sequence into a cell-free system to synthesize the structure monomer comprising the target protein. The application also provides a method for synthesizing a topological protein in a cell-free system. The purpose of the application is to provide a method for synthesizing a protein which is simpler and faster than a cell system, has an unprecedented design freedom in an open environment, and is controllable in polymerization degree and length of the synthesized protein.

Description

Technical Field

[0001] This application relates to the field of synthetic biology, specifically to a method for synthesizing a cell-free system topological protein and its structural monomers. Background Technology

[0002] Chemical tools have laid the foundation for protein topology engineering. Chemical tools used for protein synthesis include click chemistry, expression-based protein linking methods, and split-intein techniques. Click chemistry is very useful for bioconjugation, but this method can be limited by limited polymerization or a lack of sequence control. Expression-based protein linking methods utilize intein domains that form thioesters to facilitate subsequent linking / cyclization. Split-intein techniques provide opportunities for linking within the cell. The short peptide intein-C and its protein chaperone intein-N can be recombinated and spliced ​​in situ. SpyTag / SpyCatcher chemistry represents a powerful toolbox. SpyTag is a short peptide that forms a heteropeptide spy catcher when it encounters its protein chaperone. Various topological proteins, such as cyclic elastin-like peptides (ELPs), β-lactamases and dihydrofolate reductases (DHFRs), luciferases, lichenases, and 3-arm / 4-arm astrocytes and H-branched ELPs, have been successfully synthesized.

[0003] Spontaneous isopeptide bonds typically form in Gram-positive bacteria, and these are commonly found in the immunoglobulin-like domains CnaB1 and CnaB2. In Streptococcus pyogenes, isopeptide bonds can form between aspartic acid 117 (Asp117) and lysine (Lys31) of CnaB2, a fibronectin-binding protein. CnaB2 can be divided into a 13-residue peptide Spytag and a 116-residue complementary domain Spycatcher. SpyTag / SpyCatcher is a multi-subunit artificial complex protein. SpyCatcher and SpyTag can bind to other functional proteins and then act as a medium connecting the two proteins, providing a plug-and-play universal technique for constructing protein complexes. The Spytag / Spycatcher interaction is characterized by autocatalysis, rapid reaction, high yield, high thermal stability, mechanical stability, and resistance to proteases. This complex protein structure is highly advantageous for designing complex protein assemblies and constructing stable protein structures. The stable structure of Spytag / Spycatcher gives it excellent resistance to high temperatures and inhibitors. While Spytag / Spycatcher offers many advantages, its traditional preparation method involves intracellular expression followed by purification, which has limitations. For example, the expression level may not reach the desired level due to cellular space constraints, and the formation process is difficult to control. Therefore, developing a freely regulated technique is essential. Summary of the Invention

[0004] In view of the above-mentioned deficiencies of the prior art, the purpose of this application is to provide a simpler and faster protein synthesis method than the cell system, which has unprecedented design freedom in an open environment and the degree of polymerization and length of the synthesized protein can be controlled.

[0005] This application provides the following technical solution.

[0006] This application provides a method for synthesizing topological protein monomers in a cell-free system, comprising the following steps:

[0007] Synthesize a basic sequence containing a gene sequence expressing a target protein, a gene sequence expressing a SpyTag, or a gene sequence expressing a SpyCatcher.

[0008] The basic sequence was added to a cell-free system to synthesize structural monomers containing the target protein.

[0009] This application also provides a method for synthesizing topological proteins in a cell-free system, comprising the following steps:

[0010] The first basic sequence is synthesized, which contains the gene sequence expressing the target protein and the gene sequence expressing the SpyTag.

[0011] Synthesize a second basic sequence containing the gene sequence expressing the target protein and the gene sequence expressing SpyCatcher;

[0012] The first and second basic sequences were added to a cell-free system to synthesize a first structural monomer comprising SpyTag and the target protein, and a second structural monomer comprising SpyCatcher and the target protein.

[0013] The first and second structural monomers are further polymerized into a target protein with a topological structure.

[0014] Furthermore, the first and second structural monomers are purified in a cell-free system before being used to synthesize the target protein with the desired topological structure; or

[0015] The first and second structural monomers were used to directly synthesize the target protein with a topological structure in situ in a cell-free system.

[0016] Furthermore, the basic sequence also includes a gene sequence expressing a tag sequence, wherein the tag sequence is a His-tag.

[0017] Furthermore, the tag sequence is located at the C-terminus or N-terminus of the target protein gene sequence, preferably at the C-terminus.

[0018] Furthermore, the basic sequence is selected from one or more of the following: His-PS-SC, PS-SC-His, His-SC-PS, SC-PS-His, His-PS-ST, PS-ST-His, His-ST-PS, ST-PS-His, His-SC-PS-SC, SC-PS-SC-His, His-ST-PS-ST, and ST-PS-ST-His.

[0019] SpyTag is abbreviated as ST, SpyCatcher as SC, His-Tag as His, and the target protein gene sequence as PS.

[0020] Furthermore, the method for preparing the cell-free system includes the following steps: breaking the cells to obtain cell extracts, and then adding RNA polymerase and cofactors to obtain the cell-free system.

[0021] Furthermore, the cofactor includes magnesium ions, and the concentration of magnesium ions in the cell-free system is 5-35 mM, preferably 10-30 mM.

[0022] Furthermore, the RNA polymerase is a T7 RNA polymerase, preferably a T7 RNA polymerase extracted from E. coli BL21 containing plasmid pAR1219.

[0023] Furthermore, the basic sequence exists in the form of plasmids or linear DNA.

[0024] Furthermore, the target protein of the topological structure is a dimer, trimer, or multimer.

[0025] Furthermore, the molar ratio of the second basic sequence to the first basic sequence is (1-81):9, preferably (9-21):9.

[0026] The method provided in this application utilizes a cell-free system and combines SpyCatcher / SpyTag chemistry to develop a cell-free one-pot transcription-translation-assembly system for synthesizing flexibly regulated topological protein materials. Twelve basic sequences (plasmids) capable of expressing different proteins were designed and successfully expressed using the cell-free system. First, the solubility of the expressed proteins in the cell-free system was verified using polyacrylamide gel electrophoresis, laying the foundation for subsequent protein polymerization. Then, it was found that the protein with a His-tag at the C-terminus showed a higher expression level compared to plasmids with the His-tag located at the N-terminus. Next, the cell-free system was optimized by adjusting the magnesium ion concentration and the molar ratio of different plasmids to obtain the maximum degree of polymerization.

[0027] The method provided in this application enables the simple, rapid, and efficient production of proteins with controllable degree of polymerization and length through a cell-free synthesis system. This application utilizes cell extracts or enzyme systems related to transcription and translation, and co-expresses these proteins in vitro by adding energy systems, DNA templates (plasmids), salt ions, cofactors, amino acids, etc., thereby obtaining the target polymeric protein in a single batch, saving time and improving efficiency. Attached Figure Description

[0028] Figure 1 The images show the SDS-PAGE results of the supernatant and whole protein samples of the monomeric proteins prepared in Examples 1-12.

[0029] Figure 2 The fluorescence patterns of the monomeric proteins prepared in Examples 1-12 were obtained using an enzyme-linked immunosorbent assay (ELISA) reader.

[0030] Figure 3 The Western Blot results are shown for the monomeric proteins prepared in Examples 1-12.

[0031] Figure 4 The Western Blot results are shown for the preparation of polymeric proteins in Examples 13-33.

[0032] Figure 5 The Western Blot results are shown for the preparation of polymeric proteins in Examples 34-54.

[0033] Figure 6 Western blotting and fluorescence results of the polymers prepared in Examples 55-58.

[0034] Figure 7 The Western Blot results are shown for the preparation of polymeric proteins in Examples 59-62. Detailed Implementation

[0035] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0036] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0037] Protein topology has become an important dimension of protein-based materials. The chemical topology of the protein backbone offers numerous functional benefits, such as structural stability, dynamic switching properties, co-valent effects, and quaternary structural control. Protein topology engineering provides many potential functional advantages, even without altering the native sequence of the target protein. This makes protein engineering significant for constructing highly stable large complexes. However, despite the unparalleled control over sequence, length, and stereochemistry in protein biosynthesis, the diversity of protein chemical topology remains largely unexplored.

[0038] The topological features of proteins are the result of millions of years of natural evolution. Even so, nature has only explored a small fraction of all possible protein topologies. To fully realize the potential of protein topology engineering, it is wise to begin by mimicking the processes by which nature creates topological features. The awe-inspiring complexity of proteins obscures the simplicity of their design and synthesis. While transforming linear chains into loops, tadpoles, stars, and branched proteins can be as simple as a single-step joining, designing a nontrivial protein topology from scratch is extremely difficult. It requires not only precise control over protein folding but also precise control over post-translational modifications such as cleavage and joining. Therefore, creative methods are needed to achieve these complex topologies, especially those with mechanical bonds, to realize the goal of "weaving" protein chains on demand.

[0039] This application provides a method for synthesizing topological protein monomers in a cell-free system, comprising the following steps:

[0040] Step 1: Synthesize a basic sequence containing the gene sequence expressing the target protein, the gene sequence expressing SpyTag, or the gene sequence expressing SpyCatcher.

[0041] Step 2: The basic sequence is added to a cell-free system to synthesize a structural monomer containing the target protein.

[0042] Specifically, the basic sequence containing the gene sequence expressing the target protein and the gene sequence expressing the SpyTag is the first basic sequence.

[0043] The first basic sequence can be added to the cell-free system to synthesize a structural monomer containing the target protein, which is the first structural monomer.

[0044] Specifically, the basic sequence containing the gene sequence expressing the target protein and the gene sequence expressing SpyCatcher is the second basic sequence.

[0045] The addition of the second basic sequence to the cell-free system can synthesize a structural monomer containing the target protein, which is the second structural monomer.

[0046] This application provides a method for synthesizing topological proteins in a cell-free system, comprising the following steps:

[0047] Step 1: Synthesize the first basic sequence containing the gene sequence expressing the target protein and the gene sequence expressing the SpyTag;

[0048] Synthesize a second basic sequence containing the gene sequence expressing the target protein and the gene sequence expressing SpyCatcher;

[0049] Step 2: Add the first and second basic sequences to a cell-free system to synthesize a first structural monomer including SpyTag and the target protein, and a second structural monomer including SpyCatcher and the target protein.

[0050] The first and second structural monomers are further polymerized into a target protein with a topological structure.

[0051] Specifically, the first and second structural monomers are purified in a cell-free system and then the target protein with the topological structure is synthesized; or

[0052] The first and second structural monomers were used to directly synthesize the target protein with a topological structure in situ in a cell-free system.

[0053] In this application, both the first basic sequence and the second basic sequence further include a gene sequence expressing a tag sequence, wherein the tag sequence is a His-tag.

[0054] This application places no restrictions on the types of the first and second basic sequences, as long as they can serve as gene templates for synthesizing the target protein. For example, they can be linear DNA or mRNA, circular DNA or RNA, DNA or RNA of any origin, and DNA or RNA of any structural form, as long as they can serve as gene templates for eventual transcription and translation into protein. Specifically, if it is plasmid DNA, its expression plasmid only needs to carry the gene expressing the target protein.

[0055] This application does not impose any restrictions on the number of amino acids contained in the target protein to be synthesized. For example, the target protein to be synthesized may contain 10-3000 amino acids, preferably 10-1000 amino acids. For example, the target protein to be synthesized may contain 10 amino acids, 50 amino acids, 100 amino acids, 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids, 600 amino acids, 700 amino acids, 800 amino acids, 900 amino acids, 1000 amino acids, 2000 amino acids, 3000 amino acids, etc.

[0056] This application has no restrictions on the gene encoding the target protein to be synthesized. For example, it can be a gene encoding a fluorescent protein, a gene encoding a biocatalytic enzyme, a gene encoding a vaccine protein, a gene encoding an antibody protein, a gene encoding a membrane protein, a gene encoding a polypeptide, etc.

[0057] SpyTag / SpyCatcher is a multi-subunit artificial complex protein, in which SpyTag is a short peptide that forms an isopeptide bond when it encounters its protein chaperone, SpyCatcher. SpyCatcher and SpyTag can bind to other functional proteins and then act as a mediator connecting the two proteins. The SpyTag / SpyCatcher interaction is characterized by autocatalysis, rapid reaction, high yield, high thermal stability, mechanical stability, and resistance to proteases.

[0058] His-Tag is a protein purification tag. Its inherent characteristics do not affect the target protein, it does not form dimers, has a small molecular weight, does not affect downstream applications of the protein, and has low immunogenicity. The purified protein can be directly injected into animals for immunization and antibody preparation. It is compatible with bacterial transcription and translation mechanisms, which is beneficial for protein expression. It can be used to construct dual-tag expression systems with other tags, and can be used in various protein expression systems. The purification conditions are mild and have minimal impact on the protein.

[0059] In this application, the tag sequence is located at the C-terminus or N-terminus of the target protein gene sequence, preferably at the C-terminus.

[0060] In this application, the basic sequence is selected from two or more of the following: His-PS-SC, PS-SC-His, His-SC-PS, SC-PS-His, His-PS-ST, PS-ST-His, His-ST-PS, ST-PS-His, His-SC-PS-SC, SC-PS-SC-His, His-ST-PS-ST, and ST-PS-ST-His.

[0061] SpyTag is abbreviated as ST, SpyCatcher as SC, His-Tag as His, and the target protein gene sequence as PS.

[0062] Specifically, the first basic sequence is one or more of His-PS-ST, PS-ST-His, His-ST-PS, ST-PS-His, His-ST-PS-ST, or ST-PS-ST-His.

[0063] The second basic sequence can be one or more of His-PS-SC, PS-SC-His, His-SC-PS, SC-PS-His, His-SC-PS-SC, or SC-PS-SC-His.

[0064] In the synthesis of the target protein, the molar ratio of the second basic sequence to the first basic sequence is (1-81):9.

[0065] For example, the possible ratios are 1:9, 2:9, 3:9, 4:9, 5:9, 6:9, 7:9, 8:9, 9:9, 10:9, 11:9, 12:9, 13:9, 14:9, 15:9, 16:9, 17:9, 18:9, 19:9, 20:9, 21:9, 22:9, 23:9, 24:9, 25:9, 26:9, 27:9, 28:9, 29:9, 30:9, 31:9, 32:9, 33:9, 34:9, 35:9, 36:9, 37:9, 38:9, 39:9, 40:9, and 41:9. 42:9, 43:9, 44:9, 45:9, 46:9, 47:9, 48:9, 49:9, 50:9, 51:9, 52:9, 53:9, 54:9, 55:9, 56:9, 57:9, 58:9, 59:9, 60:9, 61:9, 62:9, 63:9, 64:9, 65:9, 66:9, 67:9, 68:9, 69:9, 70:9, 71:9, 72:9, 73:9, 74:9, 75:9, 76:9, 77:9, 78:9, 79:9, 80:9 or 81:9.

[0066] In this application, the target protein having a topological structure is a dimer, trimer, or multimer.

[0067] Specifically, when it is necessary to synthesize a dimer, the second basic sequence His-PS-SC, PS-SC-His, His-SC-PS and SC-PS-His are synthesized in a cell-free system with any one or more of the first basic sequence His-PS-ST, PS-ST-His, His-ST-PS or ST-PS-His, respectively. Preferably, the second basic sequence is SC-GFP-His and the first basic sequence is GFP-ST-His, and the dimer is synthesized in a cell-free system with a molar ratio of the second basic sequence to the first basic sequence of 21:9.

[0068] Specifically, when it is necessary to synthesize a trimer, the second basic sequence His-SC-PS-SC and SC-PS-SC-His are synthesized in a cell-free system with any one or more of the first basic sequences His-PS-ST, PS-ST-His, His-ST-PS, or ST-PS-His, respectively. Preferably, the second basic sequence is SC-GFP-His and the first basic sequence is ST-GFP-ST-His, and their molar ratio is 21:9, or the second basic sequence is SC-GFP-SC-His and the first basic sequence is GFP-ST-His, and their molar ratio is 9:9, and the trimer is synthesized in a cell-free system.

[0069] Specifically, when it is necessary to synthesize a trimer, the first basic sequence His-ST-PS-ST and ST-PS-ST-His are respectively synthesized with any one or more of the second basic sequences His-PS-SC, PS-SC-His, His-SC-PS or SC-PS-His in a cell-free system to synthesize a trimer. Preferably, the second basic sequence is SC-GFP-SC-His and the first basic sequence is ST-GFP-ST-His, and their molar ratio is 21:9, and a multimer is synthesized in a cell-free system.

[0070] Specifically, when it is necessary to synthesize a multimer, the first basic sequence His-ST-PS-ST and ST-PS-ST-His are respectively combined with any one or more of the second basic sequences His-SC-PS-SC or SC-PS-SC-His and / or His-PS-SC, PS-SC-His, His-SC-PS, SC-PS-His, His-PS-ST, PS-ST-His, His-ST-PS or ST-PS-His to synthesize a multimer in a cell-free system.

[0071] In this application, the method for preparing the cell-free system includes the following steps: breaking cells to obtain cell extracts, and then adding RNA polymerase and cofactors to obtain a cell-free system, that is, the cell-free system includes cell extracts, RNA polymerase and cofactors.

[0072] The cell extract is derived from bacterial cells, rabbit reticulate cells, wheat germ, or insects, preferably bacterial cell extracts; the bacterial cells can be cell extracts of any bacterial strain, such as Escherichia coli.

[0073] RNA polymerases that recognize promoters that are operatively linked to a basic sequence, such as T7 RNA polymerase;

[0074] During cell-free synthesis, the cofactors provide the substances required for protein synthesis, including, for example, energy sources, amino acids, salts, and magnesium ions (Mg). 2+ ) and other reagents.

[0075] The energy source is a chemical substrate that can be enzymatically acted to provide energy for the desired chemical reaction. Commonly used energy sources allow for the release of energy for synthesis by breaking high-energy phosphate bonds, such as those present in nucleoside triphosphates (e.g., ATP). Any source that can convert high-energy phosphate bonds is particularly suitable. Generally, ATP, GTP, and other phosphates are considered equivalent energy sources for supporting protein synthesis. In this application, a nucleoside triphosphate mixture (NTPmix) and phosphoenolpyruvate (PEP) are preferred. The nucleoside triphosphate mixture includes spermidine, putrescine, nicotinamide adenine dinucleotide, ATP, CTP, GTP, UTP, CoA, tRNA, and folinic acid.

[0076] In this application, the concentration of magnesium ions in the cell-free system is 5-50 mM. For example, the values ​​can be 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, or 50mM.

[0077] Specifically, the amino acids include arginine (Arg), valine (Val), tryptophan (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), cysteine ​​(Cys), methionine (Met), alanine (Ala), asparagine (Asn), aspartic acid (Asp), glycine (Gly), glutamine (Gln), lysine (Lys), proline (Pro), serine (Ser), threonine (Ser), and tyrosine (Tyr);

[0078] Specifically, the salt includes potassium glutamate, ammonium glutamate, and potassium oxalate monohydrate;

[0079] Specifically, the other reagents include oxidized glutathione, reduced glutathione, and PEG8000.

[0080] Specifically, the basic sequence exists in the form of plasmids or linear DNA.

[0081] The method provided in this application utilizes a cell-free system, combined with SpyCatcher / SpyTag chemistry, to develop a cell-free one-pot transcription-translation-assembly system for synthesizing flexibly regulated topological protein materials. Twelve basic sequences (plasmids) capable of expressing different proteins were designed and successfully expressed using the cell-free system. First, the solubility of the expressed protein in the cell-free system was verified by polyacrylamide gel electrophoresis, laying the foundation for subsequent protein polymerization. Then, it was found that the protein with the His-tag at the C-terminus had a higher expression level than the plasmid with the His-tag located at the N-terminus. Next, the cell-free system was optimized by adjusting the magnesium ion concentration and the molar ratio of different plasmids to obtain the maximum degree of polymerization. The results showed that the optimal magnesium ion concentration for expressing SpyCatcher / SpyTag in the cell-free system of this experiment was 10-30 mM, and the molar ratio of the first basic sequence to the second basic sequence was (1-81):9, especially (9-21):9. Different polymers had different optimal plasmid molar ratio ranges: dimer (3-8):2, trimer (4-24):6, and multimer (3-7):3.

[0082] Example

[0083] The materials and test methods used in the embodiments of this application are described in a general and / or specific manner. In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0084] Preparation of components of cell-free synthetic system

[0085] A. Preparation of cell extracts

[0086] (1) The buffer solutions used for cell extracts are shown in Table 1:

[0087] Table 1 Buffer solutions required for cell extract preparation

[0088]

[0089] (2) Bacterial culture: Single colonies were picked from antibiotic-free 2×YT solid medium and transferred to 2×YT liquid medium, and cultured overnight with primary seed culture. The primary seed culture was added to fresh 2×YT liquid medium at a 5% inoculum volume, and cultured until the cells reached the middle of the logarithmic growth phase to prepare secondary seed culture. The secondary seed culture was transferred to 4×1L medium at a 5% inoculum volume for cell growth. At the end of the logarithmic growth phase, the bacterial culture was collected, centrifuged at 8000 rpm, 4℃, for 30 min, and the supernatant was discarded.

[0090] (3) Resuspend the bacteria in the disruption buffer, then centrifuge at 8000 rpm, 4℃, and 10 min. Repeat twice and weigh the bacteria.

[0091] (4) Add 1 mL of lysis buffer to each gram of bacterial cells, resuspend, and place on ice.

[0092] (5) Before grinding with a high-pressure grinder, an ice-water mixture covering the grinding section must be added to prevent the high temperature during grinding from affecting the activity of the extract. Turn on the high-pressure grinder and control the pressure at 1000 bar. After the ethanol flows out of the injection cup, add two columns of sterile water, then add one column of lysis buffer. After the lysis buffer flows out, add the resuspended bacterial solution for grinding, and repeat the grinding operation. Centrifuge the ground sample at 10,000 rpm and 4°C for 30 minutes. Damaged injection cups need to be cleaned with two columns of sterile water and two columns of 75% ethanol, and finally sealed with 75% ethanol.

[0093] (6) Transfer the supernatant after centrifugation to a new sterile BD tube, wrap it with aluminum foil, and incubate it on a shaker at 120 rpm at 37°C for 80 minutes in the dark.

[0094] (7) Centrifuge the sample at 10,000 rpm for 30 minutes at 4℃.

[0095] (8) Aspirate the supernatant and add it to a 6-8 kDa MWCO dialysis bag. Dialyze overnight at 4°C in dialysis buffer.

[0096] (9) Place the cell extract from the dialysis bag into a BD tube, 10000 rpm, 4℃, for 30 minutes, then aliquot the supernatant into multiple 1.5 mL EP tubes, freeze quickly with liquid nitrogen, and store in a -80℃ freezer.

[0097] B. Preparation of T7 RNA polymerase

[0098] Prepared using E. coli BL21(DE3) strain chemically transformed with pAR1219 plasmid.

[0099] (1) The required buffer solutions are prepared as shown in Table 2:

[0100] Table 2. Components of the buffer solution required for the preparation of T7 RNA polymerase in this paper.

[0101]

[0102] (2) Activation of strain: The E. coli BL21(DE3)-PAR1219 strain stored at -80℃ was aspirated onto ampicillin LB medium and cultured at 37℃ for 12h in a constant temperature incubator;

[0103] (3) Pick a single colony and transfer it to LB liquid medium (20% liquid content), 200 rpm, 37 ℃, constant temperature shaker for 12 h;

[0104] (4) Transfer the bacterial culture to 1L of fresh LB liquid medium with an inoculation amount of 5% and incubate at 200rpm and 37℃ in a constant temperature shaker for about 1 hour;

[0105] (5) When the OD600 is 0.6 to 0.8, add 1 mM IPTG (1%);

[0106] (6) When OD600 is 2.0, centrifuge at 8000 rpm, 4℃, for 20 min and collect cells;

[0107] (7) Resuspend the cells in pre-cooled S30 buffer at a ratio of 5 mL / g cells and centrifuge at 8000 rpm for 20 minutes at 4°C. Repeat this process twice;

[0108] (8) Resuspend the cells in lysis buffer at a ratio of 4 mL / g cells, sonicate for 40 min (working for 2 s, resting for 6 s), and change the ice every 10 min.

[0109] (9) Centrifuge at 8000 rpm, 4℃, for 20 min, discard the precipitate, and retain the supernatant;

[0110] (10) Add the supernatant to a 6-8 kDa MWCO dialysis bag and dialyze overnight at 4°C with dialysis buffer;

[0111] (11) Transfer the liquid in the dialysis bag to the BD tube, centrifuge at 10,000×g, 4℃ for 30min, take the supernatant, dispense it onto ice, freeze the liquid quickly and store it in a -80℃ freezer.

[0112] C. Preparation of cell-free partial solutions

[0113] Table 3. Composition of some solutions in the cell-free system in this experiment.

[0114]

[0115] D. Preparation of a 25× mixed solution of 19 amino acids

[0116] All 19 amino acids were prepared at a concentration of 50 mM. During preparation, each amino acid was completely dissolved before adding the next. Tyrosine, added last, was insoluble. After thorough mixing, the pH was adjusted to 7.4 with concentrated hydrochloric acid. The composition is shown in Table 4.

[0117] Table 4. Components of the 19 amino acid mixture in the cell-free system 25× in this experiment.

[0118]

[0119] Material characterization

[0120] A. Microplate reader fluorescence test

[0121] This study used a TECAN M200P40 microplate reader. Fluorescent proteins were diluted 20-40 times with 1×PBS to measure fluorescence intensity. The microplate reader was shaken for 30 seconds before measurement to ensure thorough mixing. All proteins in this paper contained green fluorescent protein (sfGFP), with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

[0122] B. Transmission electron microscope (TEM)

[0123] In this study, a FEI D1266 transmission electron microscope was used. First, 5 μL of a sample diluted 100-fold was dropped onto an ultrathin carbon-supported film. 5 μL of uranium acetate (2%) was added to the film, allowed to stand for 1 minute, excess uranium acetate was removed with filter paper, and the film was allowed to air dry for 1 minute. Finally, the treated ultrathin carbon-supported film was observed in a TEM at 120 kV.

[0124] C. Laser confocal microscope

[0125] In this study, a Zeiss LSM-780 laser confocal microscope was used. First, the sample was diluted 100-fold and then dropped onto a glass slide with coverslips. The confocal microscope was adjusted to the excitation and emission wavelengths of the corresponding fluorescent protein. After locating the sample under a 10x objective, the microscope was switched to a 60x oil objective to observe the morphology of the fluorescent protein.

[0126] Monomeric protein expression

[0127] Example 1

[0128] To explore the synthesis of topological proteins in a cell-free system, green fluorescent protein (GFP) was used as a model. The gene sequences of GFP, His-tag, SpyTag, and SpyCatcher were obtained from NCBI (sequences shown in Table 5), and ligation was performed using Snapgene with the ligation sequence GGGGSGGGGS. The constructed gene sequences were embedded into the pET23a vector, with Nde1 and HindIII restriction enzyme sites, and ordered from Kingwise Biotech Co., Ltd. The pET23a vector was used. DH5α stab bacteria for plasmid extraction were provided by Kingwise Biotech. Single colonies were obtained by streaking solid LB medium in three zones. Each single colony was inoculated into 10 mL of LB medium containing 100 μg / mL ampicillin antibiotic at 37°C. Both streaking and inoculation were performed in a clean bench, and the medium was incubated overnight on a shaker at 37°C. The cells were harvested by centrifugation the next day, and plasmids were extracted using the Qiagen PlasmidMaxi Kit (Qiagen, Valencia, CA) to obtain the basic sequence.

[0129] Table 5

[0130]

[0131]

[0132] Add the components listed in Table 6 below sequentially to 1.5 mL EP tubes, with a total reaction volume of 20 μL. Make up any remaining fraction (less than 20 μL) with sterile water. Incubate at 30°C for 12 hours in a biochemical incubator. After incubation, collect the whole protein and verify expression by measuring the fluorescence value of the fluorescent protein using SDS-PAGE, Western blotting, and a microplate reader. Results are as follows... Figures 1-3 As shown.

[0133] Table 6

[0134]

[0135] Examples 2-12 differ from Example 1 in their basic sequences, as shown in Table 7, and the results are as follows. Figures 1-3 As shown.

[0136] Table 7

[0137]

[0138] Summary: From the table above and Figures 1-3SDS-PAGE analysis of the whole protein solution and supernatant after centrifugation of the cell-free products revealed that the designed monomeric proteins were synthesized in Examples 1-12 in the cell-free system, and all 12 designed monomeric proteins were soluble. Soluble proteins are more easily dispersed in the cell-free system, which is more conducive to subsequent polymerization and lays a good foundation and conditions for subsequent protein polymerization. Moreover, the protein expression level was higher when the His-tag was located at the C-terminus. It is speculated that the N-terminal His tag sequence may affect protein translation, especially the ST fusion protein. The expression level of SC-GFP-His(C) in the cell-free system was 15 times that of (N)His-SC-GFP. Compared with (N)His-GFP-ST, GFP-ST-His(C) increased by 8 times. Compared with (N)His-ST-GFP-ST, ST-GFP-ST-His(C) increased by 5 times. The only opposing result was that (N)His-SC-GFP-SC had a higher fluorescence value than SC-GFP-SC-His(C). Western blot analysis revealed that SC-GFP-SC-His(C) was expressed at a higher rate than (N)His-SC-GFP-SC. It is speculated that GFP may be encapsulated by SpyCatcher, which weakens the green fluorescence, leading to the lower fluorescence value. Although different constructs showed different CFPS results, they were successfully expressed.

[0139] expression of topogen

[0140] Example 13

[0141] Take 5 μL of each of the liquids prepared in Example 6 (containing sfGFP-ST-His(I)) and Example 4 (containing SC-sfGFP-His(H)) and place them in new 1.5 mL EP tubes. Incubate at room temperature for three hours. Stable isopeptide bonds can spontaneously form between SpyCatcher and SpyTag, allowing for a complete reaction to obtain the maximum amount of polymerized protein. Western blotting and ImageJ analysis were performed to calculate the polymerization ratio. Figure 4 .

[0142] from Figure 4 As can be seen from the data, after the reaction, the cell-free system contained both synthesized dimers and unreacted structural monomers. The proportion of dimers formed was 81%, and their size was 70 kDa. The parameters are shown in Table 8.

[0143] The difference between Examples 14-33 and Example 13 lies in the different combination methods of the structural units. The parameters are shown in Table 8. Figure 4 .

[0144] Table 8

[0145]

[0146]

[0147] Summary: Based on the table above and Figure 4 It is known that by constructing the second and first structural monomers using SpyCatcher and SpyTag, and adding equal volumes of the first and second structural monomers to a cell-free synthesis system, the first and second structural monomers were polymerized, and proteins with topological structures, including dimers, trimers, and multimers, were successfully synthesized.

[0148] In situ co-expression and aggregation

[0149] Example 34

[0150] First, a cell-free system containing two different plasmids was established. 6 μL of cell extract, 0.2 μL of 1 mol / L magnesium glutamate, 0.8 μL of 19AA, 1 μL of Hepes buffer, 0.8 μL of NTPs, 0.8 μL of LPEP, 0.1 μL of L7 RNA polymerase, and 1 μL of 10×salt were placed in a 1.5 mL EP tube. The molar number of the first and second basic sequences was 5 × 10⁻⁶. -12 The solution was added to mol, then diluted to 20 μL with sterile water. The mixture was vortexed and incubated overnight at 30°C. The resulting green fluorescent polymer was obtained. Stable heteropeptide bonds spontaneously form between SpyCatcher and SpyTag, allowing for a complete reaction to maximize the polymer's yield. After co-expression, the proteins were classified as dimers, trimers, and multimers and spotted onto the same SDS-Page gel. Western blot analysis and ImageJ analysis were performed to calculate the polymerization ratio. Figure 5 Its parameters are shown in Table 9.

[0151] The difference between Examples 35-54 and Example 34 lies in the different combinations of the basic sequences, as detailed in Table 9. Figure 5 .

[0152] Table 9

[0153]

[0154] Summary: Based on the table above and Figure 5 It is evident that by adding the basic sequence constructed using SpyCatcher and SpyTag to a cell-free system, proteins with topological structures, including dimers, trimers, and multimers, were successfully synthesized, achieving in situ co-expression and promoting the aggregation of complex proteins.

[0155] The effect of magnesium ions in cell-free systems

[0156] Example 55

[0157] Thirteen cell-free systems were constructed, containing increasing magnesium ion concentrations (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, and 200 mmol / L). First, the components listed in Table 6, excluding magnesium ions, were added sequentially to 1.5 mL EP tubes. Then, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, and 4.0 μL of 1 mol / L magnesium ion solution were added. The first and second basic sequences were then added, selected according to the combination method in Table 10, with each set of the first and second basic sequences having a molar number of 5 × 10⁻⁶. -12 The total reaction volume was 20 μL, and any fraction less than 20 μL was made up with sterile water. The reaction was carried out at 30°C for 12 hours in a biochemical incubator. The magnesium ion concentration of the maximum polymer was then determined by Western blotting and ImageJ analysis, as shown in the results. Figure 6 The parameters are shown in Table 10.

[0158] The difference between Examples 56-58 and Example 55 lies in the combination method of the basic sequence. The parameters are shown in Table 10, and the results are as follows: Figure 6 .

[0159] Table 10

[0160]

[0161] In summary, different magnesium ion concentrations did indeed affect cell-free expression and polymerization in the four combinations, such as... Figure 6 As shown. Figure 6 As shown in A1, B1, C1, and D1, when Mg 2+ When the ion concentration is 0, monomeric proteins cannot be expressed, which further confirms the importance of magnesium ions. From Figure 6 From D1 and D2, we can see that, regarding the synthesis of the polymer K+L, when Mg... 2+ At a concentration of 5 mM ions, monomers were expressed to some extent, but failed to form polymers. It is speculated that Mg... 2+ Ions also have a certain influence on protein polymerization. When Mg in a cell-free system... 2+ When the ion concentration is above 10 mM, dimers, trimers, and polymers can be formed. Those skilled in the art can choose the appropriate type based on their needs and synthesis requirements. For example, when Mg... 2+At ion concentrations of 10–30 mM, the expression levels of dimers, trimers, and polymers were relatively high. The maximum formation ratios of dimers, trimers, and polymers reached 1.0, 0.54, and 0.29, respectively.

[0162] The effect of the molar ratio of basic sequences (plasmids) in cell-free systems

[0163] Example 59

[0164] Nine cell-free systems were designed, with molar ratios of the second basic sequence to the first basic sequence of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, respectively. The total molar ratio of the first and second basic sequences was 10 × 10⁻⁶. -12 Based on the above optimization results of magnesium ion concentration, a magnesium ion concentration of 15 mmol / L will be used in the following experiments. The total reaction volume is 20 μL. After adding all the components in Table 6, any remaining less than 20 μL will be made up with sterile water. The reaction conditions are 30℃ and 12 h in a biochemical incubator. Then, Western blotting and ImageJ analysis will be used to obtain the plasmid molar ratio of the largest polymer for different polymers. The results are as follows: Figure 7 The parameters are shown in Table 11, and the results are as follows: Figure 7 .

[0165] The difference between Examples 60-62 and Example 59 lies in the combination method of the basic sequence. The parameters are shown in Table 11, and the results are as follows: Figure 7 .

[0166] Table 11

[0167]

[0168]

[0169] Summary: From Figure 7 It can be seen that different plasmid molar ratios revealed different polymerization results. The optimal polymerization molar ratio for the dimer (SC-GFP-His+GFP-ST-His, H+I) was (3-8):2, for the trimer (SC-GFP-His+ST-GFP-ST-His, H+L) it was (3-8):2, for the trimer (SC-GFP-SC-His+GFP-ST-His, K+I) it was (4-9):6, and for the multimer (SC-GFP-SC-His+ST-GFP-ST-His, K+L) it was (3-7):3. The final proportion of dimers reached 73%, trimers reached 53% or 60%, and multimers reached 36%.

[0170] Although the embodiments of this application have been described above in conjunction with the accompanying drawings, this application is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, not restrictive. Those skilled in the art can make many other forms based on the guidance of this specification and without departing from the scope of protection of the claims of this application, and these are all within the scope of protection of this application. sequence list <110> Tsinghua University <120> A method for synthesizing cell-free system topological proteins and their structural monomers. <130> TPF02178 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesized sequences <400> 1 gcccacattg tgatggtgga tgcctataag ccaacaaaat aa 42 <210> 2 <211> 339 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesized sequences <400> 2 gtagacaccc tgagtggcct cagcagtgaa caaggccaaa gtggagatat gaccatgaa 60 gaagatagtg ccactcatat caaattctcc aaaagagatg aagatggcaa agaactggct 120 ggtgccacta tggaactgag agatagcagt ggcaaaacca tctcaacctg gattagtgat 180 ggccaagtga aggattttta tctataccct ggcaaatata cctttgtgga aactgcagcc 240 cctgatggct atgaagtagc cacagccatt actttcacag ttaatgaaca aggccaagtt 300 acagtcaatg gcaaagccac caaaggggat gctcatatt 339 <210> 3 <211> 711 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: The sequence is artificially synthesized <400> 3 agtaagggtg aagaactgtt tactggtgtg gtgccaattc tggtggaact ggatggtgat 60 gtgaatggcc ataaatttag tgtgagaggt gaaggtgaag gtgatgccac caatggcaaa 120 ctgaccctga aatttatttg caccactggc aaactgcctg tgccatggcc aaccctggtg 180 accaccctga cctatggtgt gcagtgcttt agcagatatc ctgatcacat gaaaagacat 240 gattttttta aaagtgccat gcctgaaggc tatgtgcaag aaagaaccat tagctttaaa 300 gatgatggca cctataaaac tagagcagaa gtgaaatttg aaggtgatac cctggtgaac 360 agaattgaac tgaaaggcat tgattttaaa gaagatggca acattctggg ccataaactg 420 gaatataact ttaacagcca taatgtgtat attactgcag ataaacagaa aaatggcatt 480 aaagccaact ttaaaattag acataatgtg gaagatggca gtgtgcagct ggcagatcat 540 tatcagcaga acacccccaat tggtgatggc cctgtgctgc tgcctgataa ccattatctg 600 agcactcaga gtgtgctgag caaagatcca aatgaaaaaa gagatcacat ggtgctgctg 660 gaatttgtga ctgcagctgg cattacccat ggcatggatg aactgtataa a 711 <210> 4 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: Synthesized sequences <400> 4 catcatcatc atcatcac 18

Claims

1. A method for synthesizing topological proteins in a cell-free system, characterized in that, Includes the following steps: The first basic sequence is synthesized, which contains the gene sequence expressing the target protein and the gene sequence expressing the SpyTag. Synthesize a second basic sequence containing the gene sequence expressing the target protein and the gene sequence expressing SpyCatcher; The first and second basic sequences were added to a cell-free system to synthesize a first structural monomer comprising SpyTag and the target protein, and a second structural monomer comprising SpyCatcher and the target protein. The first and second structural monomers are further polymerized into a target protein with a topological structure; When synthesizing the dimer, the combination of the first basic sequence and the second basic sequence is selected from one of I+H, I+B, J+H, J+G, J+B, I+G, I+A, L+A and K+C; When synthesizing the trimer, the combination of the first basic sequence and the second basic sequence is selected from one of F+B, F+G, K+I, F+H and E+J; When synthesizing a mixture of dimers and trimers, the combination of the first basic sequence and the second basic sequence is selected from one of E+J, E+I, K+J, L+B, L+H, K+C, and L+G; When synthesizing mixtures of dimers, trimers, and polymers, the combination of the first basic sequence and the second basic sequence is E+L or K+L; In this context, SpyTag is abbreviated as ST, SpyCatcher as SC, His-Tag as His, and the target protein gene sequence as PS; His-GFP-SC is abbreviated as A, GFP-SC-His as G, His-SC-GFP as B, SC-GFP-His as H, His-GFP-ST as C, GFP-ST-His as I, His-ST-GFP as D, ST-GFP-His as J, His-SC-GFP-SC as E, SC-GFP-SC-His as K, His-ST-GFP-ST as F, and ST-GFP-ST-His as L. The nucleotide sequence of ST is SEQ ID NO: 1, and the nucleotide sequence of SC is SEQ ID NO:

2.

2. The method according to claim 1, characterized in that, The first and second structural monomers were purified in a cell-free system and then the target protein with the topological structure was synthesized; or The first and second structural monomers were used to directly synthesize the target protein with a topological structure in situ in a cell-free system.

3. The method according to claim 2, characterized in that, When the first and second structural monomers are purified in a cell-free system and then the target protein with the topological structure is synthesized... When synthesizing the dimer, the combination of the first basic sequence and the second basic sequence is selected from one of I+H, I+B, J+H, J+G, J+B, I+G, I+A and L+A; When synthesizing the trimer, the combination of the first basic sequence and the second basic sequence is selected from one of F+B, F+G, K+I and F+H; When synthesizing a mixture of dimers and trimers, the combination of the first basic sequence and the second basic sequence is selected from one of E+J, E+I, K+J, L+B, L+H, K+C, and L+G.

4. The method according to claim 2, characterized in that, When the first and second structural monomers are used to directly synthesize the target protein with the topological structure in situ in a cell-free system... When synthesizing the dimer, the combination of the first basic sequence and the second basic sequence is selected from one of I+H, I+B, J+H, J+G, J+B, I+G, L+A, and K+C; When synthesizing the trimer, the combination of the first basic sequence and the second basic sequence is selected from one of F+B, F+G, F+H and E+J; When synthesizing a mixture of dimers and trimers, the combination of the first basic sequence and the second basic sequence is selected from one of E+I, K+J, K+I, L+B, L+H, and L+G.

5. The method according to claim 1, characterized in that, The preparation method of the cell-free system includes the following steps: breaking the cells to obtain cell extract, and then adding RNA polymerase and cofactors to obtain the cell-free system.

6. The method according to claim 5, characterized in that, The cofactor includes magnesium ions, and the concentration of magnesium ions in the cell-free system is 5-35 mM.

7. The method according to claim 5, characterized in that, The cofactor includes magnesium ions, and the concentration of magnesium ions in the cell-free system is 10-30 mM.

8. The method according to claim 5, characterized in that, The RNA polymerase is T7 RNA polymerase.

9. The method according to claim 8, characterized in that, The RNA polymerase is a T7 RNA polymerase extracted from E. coli BL21 containing plasmid pAR1219.

10. The method according to claim 1, characterized in that, The basic sequence exists in the form of plasmids or linear DNA.

11. The method according to claim 1, characterized in that, The molar ratio of the second basic sequence to the first basic sequence is (1~81):

9.

12. The method according to claim 1, characterized in that, The molar ratio of the second basic sequence to the first basic sequence is (9~21):9.

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