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CN-115109815-B - Cell-free system topology protein and synthesis method of structural monomer thereof

CN115109815BCN 115109815 BCN115109815 BCN 115109815BCN-115109815-B

Abstract

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

Inventors

  • LU YUAN
  • WANG TING

Assignees

  • 清华大学

Dates

Publication Date
20260512
Application Date
20220426

Claims (12)

  1. 1. A method for synthesizing a cell-free system topology protein, which is characterized by comprising the following steps: Synthesizing a first base sequence comprising a gene sequence expressing a protein of interest and a gene sequence expressing SpyTag; Synthesizing a second base sequence comprising a gene sequence expressing a protein of interest and a gene sequence expressing SpyCatcher; Adding the first base sequence and the second base sequence to a cell-free system to synthesize a first structural monomer comprising SpyTag and a target protein, and a second structural monomer comprising SpyCatcher and a target protein; the first structural monomer and the second structural monomer are further polymerized into target protein with a topological structure; when synthesizing dimers, the combination of the first base sequence and the second base 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 a 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 base sequence and the second base sequence is selected from one of e+ J, E + I, K + J, L + B, L + H, K +c and l+g; When synthesizing a mixture of dimers, trimers, and multimers, the combination of the first base sequence and the second base sequence is e+l or k+l; Wherein SpyTag is abbreviated as ST, spycatcher is abbreviated as SC, his-Tag is abbreviated as His, target protein gene sequence is abbreviated as PS, his-GFP-SC is abbreviated as A, GFP-SC-His is abbreviated as G, his-SC-GFP is abbreviated as B, SC-GFP-His is abbreviated as H, his-GFP-ST is abbreviated as C, GFP-ST-His is abbreviated as I, his-ST-GFP is abbreviated as D, ST-GFP-His is abbreviated as J, his-SC-GFP-SC is abbreviated as E, SC-GFP-SC-His is abbreviated as K, his-ST-GFP-ST is abbreviated as F, ST-GFP-ST-His is abbreviated as L, The nucleotide sequence of the ST is SEQ ID NO. 1, and the nucleotide sequence of the SC is SEQ ID NO. 2.
  2. 2. The method of claim 1, wherein the first structural monomer and the second structural monomer are purified in a cell-free system and then the target protein with a topological structure is synthesized, or The first structural monomer and the second structural monomer directly synthesize the target protein with the topological structure in situ in a cell-free system.
  3. 3. The method according to claim 2, wherein when the target protein having a topological structure is synthesized after purifying the first structural monomer and the second structural monomer in a cell-free system, When synthesizing a 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 a 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 base sequence and the second base sequence is selected from one of E+ J, E + I, K + J, L + B, L + H, K +C and L+G.
  4. 4. The method of claim 2, wherein when the first structural monomer and the second structural monomer are used to directly synthesize the target protein having a topological structure in situ in a cell-free system, When synthesizing a 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 a 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 base sequence and the second base sequence is selected from one of E+ I, K + J, K + I, L + B, L +H and L+G.
  5. 5. The method of claim 1, wherein the cell-free system is prepared by disrupting cells to obtain a cell extract, and adding RNA polymerase and cofactors to obtain the cell-free system.
  6. 6. The method of claim 5, wherein the cofactor comprises magnesium ions and the concentration of magnesium ions in the cell-free system is 5-35mM.
  7. 7. The method of claim 5, wherein the cofactor comprises magnesium ions and the concentration of magnesium ions in the cell-free system is 10-30mM.
  8. 8. The method of claim 5, wherein the RNA polymerase is a T7 RNA polymerase.
  9. 9. The method of claim 8, wherein the RNA polymerase is T7 RNA polymerase extracted using e.coli BL21 containing plasmid pAR 1219.
  10. 10. The method of claim 1, wherein the base sequence is in the form of a plasmid or linear DNA.
  11. 11. The method of claim 1, wherein the molar ratio of the second base sequence to the first base sequence is (1-81): 9.
  12. 12. The method of claim 1, wherein the molar ratio of the second base sequence to the first base sequence is (9-21): 9.

Description

Cell-free system topology protein and synthesis method of structural monomer thereof Technical Field The application relates to the technical field of synthetic biology, in particular to a cell-free system topology protein and a synthetic method of a structural monomer thereof. Background The chemical tool lays the foundation of protein topology engineering. Chemical tools for synthesizing proteins include click chemistry, expressed protein ligation methods, and split intein technology. Click chemistry is very useful for bioconjugation, but this approach may suffer from limited degree of polymerization or lack of sequence control. Expressed protein ligation methods utilize thioester-forming intein domains to facilitate subsequent ligation/cyclization. Split intein technology provides the opportunity to make links in cells. The short peptide fragment intein-C and its chaperone intein-N can be recombined and spliced in situ. SpyTag/SpyCatcher chemistry represents a powerful tool kit. SpyTag is a short peptide that forms isopeptidic bond spyware when it encounters its chaperone. Various topoproteins, such as cyclic elastin-like polypeptides (ELPs), beta-lactamases and dihydrofolate reductase (DHFR), luciferases, and lichenases, and 3-arm/4-arm astroproteins and H-branched ELPs, have been successfully synthesized. Spontaneous isopeptidic bonds are typically formed in gram-positive bacteria, such isopeptidic bonds are typically present in immunoglobulin-like domains CnaB1 and CnaB 2. An isopeptide bond may be formed between aspartic acid 117 (Asp 117) and lysine (Lys 31) of CnaB2 of the fibronectin binding protein of streptococcus pyogenes. CnaB2 can be divided into 13 residues of peptide Spytag and 116 residues of complementary domain SPYCATCHER. SpyTag/SpyCatcher is a multi-subunit artificial composite protein, and SpyCatcher and SpyTag can be combined with other functional proteins and then serve as a medium for connecting the two proteins, so that a plug-and-play universal technology is provided for constructing a protein complex. 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 very advantageous for designing complex protein assemblies and constructing stable protein structures. The stable structure of Spytag/SPYCATCHER gives it very excellent resistance to both high temperatures and inhibitors. Although spatag/SPYCATCHER has such many advantages, the conventional preparation method is to express and repurify in cells, so that there are some limitations, such as that the limited expression amount in cell space does not reach the ideal effect, the formation process is not easy to regulate, and the development of free regulation technology is very necessary. Disclosure of Invention In view of the above drawbacks of the prior art, an object of the present application is to provide a method for synthesizing a protein which is simpler and faster than a cellular system, has an unprecedented degree of freedom in design in an open environment, and allows a synthesized protein to have a controllable degree of polymerization and a controllable length. The application provides the following technical scheme. The application provides a method for synthesizing a cell-free system topological protein structural monomer, which comprises the following steps: Synthesizing a basic sequence comprising a gene sequence expressing a protein of interest, a gene sequence expressing SpyTag, or a gene sequence expressing SpyCatcher; the base sequence is added to a cell-free system to synthesize structural monomers comprising the protein of interest. The application also provides a method for synthesizing the topological protein of the cell-free system, which comprises the following steps: Synthesizing a first base sequence comprising a gene sequence expressing a protein of interest and a gene sequence expressing SpyTag; Synthesizing a second base sequence comprising a gene sequence expressing a protein of interest and a gene sequence expressing SpyCatcher; Adding the first base sequence and the second base sequence to a cell-free system to synthesize a first structural monomer comprising SpyTag and a target protein, and a second structural monomer comprising SpyCatcher and a target protein; The first structural monomer and the second structural monomer are further polymerized into a target protein with a topological structure. Further, purifying the first structural monomer and the second structural monomer in a cell-free system and then synthesizing the target protein with a topological structure, or The first structural monomer and the second structural monomer directly synthesize the target protein with the topological structure in situ in a cell-free system. Further, the basic sequence also comprises a gene sequence for expressing a tag sequence, wherein the tag sequence is Hi