Regional protein directed evolution system and method for carrying out block directed evolution on target protein

Abstract

The invention provides a protein directed evolution system based on regional search, the system comprises a to-be-directed evolution element, a gene targeted mutation element and a bacteriophage directed screening element, the to-be-directed evolution element is arranged on a bacteriophage genome vector, and the bacteriophage directed screening element of the bacteriophage genome vector is deleted; the phage directional screening element is arranged on a second vector, and the second vector is suitable for being expressed in bacteria or fungi; the expression product of the to-be-directed evolutionary element regulates and controls the expression of the phage directed screening element, and the activity of the expression product of the to-be-directed evolutionary element is in positive correlation with the expression of the phage directed screening element. The system can be used for carrying out regional directional transformation on the protein to be evolved in a high-efficiency, low-cost and multi-capture intermediate mutant manner.

Description

Technical Field

[0001] The present invention relates to the field of protein evolution, specifically, the present invention relates to a regional protein directed evolution system and a method for regional directed evolution of target proteins, more specifically, the present invention relates to a regional directed evolution system for phage-assisted gene mutation, protein expression, phenotypic selection and screening, and a method for regional directed evolution of target proteins. Background Art

[0002] Protein engineering is an important research field that involves modifying and optimizing protein structure and function. Proteins are macromolecules that are widely present in organisms and play key roles in many biological processes, including catalytic reactions, signal transduction, structural support, etc.

[0003] Protein modification is currently carried out mainly through four methods: random mutation, targeted mutation, DNA recombination, and primer amplification. Among them, random mutation is achieved through random primers, low-precision PCR, or mutant library construction methods (such as DNA disease treatment); targeted mutation (targeted point mutation, recombination, insertion, or deletion) can be precisely designed according to the target properties; DNA recombination recombines fragments or genes from different protein sources to generate new protein variants; primer amplification generates a DNA fragment library containing protein variants, and uses different primers or primer combinations to amplify the protein coding sequence through PCR reactions. However, the current protein modification methods have low efficiency and high labor costs in screening active mutants.

[0004] Therefore, it is urgent to develop a protein modification method to improve the efficiency of obtaining active mutants and reduce labor costs. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0006] The inventors found that the existing protein modification methods are inefficient, labor-intensive, and may miss some intermediate mutants that do not perform as well as the final product. To overcome this problem, the inventors constructed a highly efficient directed evolution system that, while ensuring relatively low costs, obtains a certain number of combined mutants with multi-point mutations and captures intermediate mutants in the evolution process to meet the comprehensive modification of the protein to be evolved.

[0007] In the first aspect of the present invention, the present invention proposes a sub-regional protein directed evolution system. According to an embodiment of the present invention, the system includes: an element to be directed evolution, a gene targeting mutation element and a phage directed screening element, wherein the element to be directed evolution is arranged on a phage genome vector, and the phage directed screening element of the phage genome vector is missing; the phage directed screening element is arranged on a second vector, and the second vector is suitable for expression in bacteria or fungi; the gene targeting mutation element includes a gene directed evolution tool and a gene targeting indicator element, and the gene directed evolution tool and the gene targeting indicator element are arranged on the same or different vectors, preferably on different vectors; the expression product of the gene directed evolution tool is suitable for performing sub-regional directed evolution processing on the element to be directed evolution, the gene directed evolution tool is arranged on the third vector, and the third vector is suitable for expression in bacteria or fungi; the gene targeting indicator element is arranged on the second vector; the expression product of the element to be directed evolution regulates the expression of the phage directed screening element, and the activity of the expression product of the element to be directed evolution is positively correlated with the expression of the phage directed screening element. The system according to the embodiment of the present invention can carry out region-specific directed transformation of the protein to be evolved in a manner of high efficiency, low cost and capturing multiple intermediate mutants.

[0008] In the second aspect of the present invention, the present invention proposes a method for the block directed evolution of a target protein. According to an embodiment of the present invention, the method utilizes the protein directed evolution system of the first aspect of the present invention carrying the target protein to be directed evolution to transform the target protein to be directed evolution. According to the method of the embodiment of the present invention, the protein to be evolved can be comprehensively directed transformed in a manner of high efficiency, low cost, and multiple capture of intermediate mutants.

[0009] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0011] Figure 1 This is a schematic diagram of the EvoScan system of the present invention.

[0012] Figure 2 This is the EvoScan combined mutation flow chart of the present invention (taking AmeR as an example).

[0013] Figure 3 Flow chart of a “step” in the EvoScan evolution path of the present invention.

[0014] Figure 4 This is the design of the gene circuit for the evolution of EGFP nanobody in Example 1.

[0015] Figure 5 The EvoScan process and results of the EGFP-nanoantibody interaction in Example 1 are displayed.

[0016] Figure 6 M in Example 2 pro EvoScan system construction and effect test diagram.

[0017] Figure 7 M in Example 2 pro EvoScan evolutionary design for escaping small molecule inhibition, phage proliferation and mutant summary.

[0018] Figure 8 This is the design diagram of the EvoScan evolution system for AmeR inhibition in Example 3.

[0019] Fig. 9 This is the EvoScan evolution process and result statistics of AmeR in Example 3.

[0020] Fig.10 This is a graph showing the experimental results of the induced expression of the AmeR inhibitory effect in Example 3.

[0021] Fig.11 This is the PANCE evolutionary design and result diagram of the interaction between EGFP nanoantibody and EGFP in Comparative Example 1.

[0022] Fig.12 M in Comparative Example 2 pro PANCE evolutionary design and results for escaping GC376 inhibition.

[0023] Fig.13 This is the PANCE evolution design and evolution result diagram of the AmeR inhibitory effect in comparative example 3.

[0024] Fig.14 This is the map of the MP6 mutant plasmid in Comparative Example 1.

[0025] Fig.15 This is a diagram of the reverse two-hybrid system construction and effect test of HCV protease in Example 4.

[0026] Fig.16 This is a diagram showing the effect of the reverse two-hybrid system of HCV protease in Example 4. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0028] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0029] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0030] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0031] In this article, the term "Protein Directed Evolution System" is an experimental method used to modify the function and properties of proteins. It combines the principles of protein engineering and evolutionary biology, and aims to create protein molecules with specific properties through a human-directed evolution process. The protein directed evolution system usually includes the following steps: Diversity Introduction: Create a diverse protein library by introducing random mutations or recombination. This can be achieved through gene mutations, DNA recombination or other methods; Selection or Screening: Expose the members of the protein library to specific conditions, and then select protein variants with target functions or properties through screening or selection. This can be done through high-throughput screening, functional identification or other technical means; Amplification and Repetition: Amplify the selected protein variants to increase their number and use them as the starting point for the next round of evolution. This process can be done through PCR (polymerase chain reaction) or other amplification techniques; Iterations: Repeat the steps of selection / screening, amplification and repetition to gradually optimize the performance of the protein. Each round of evolution can produce protein variants that are closer to the target function. Through multiple iterations, the protein directed evolution system can create protein molecules with specific functions or properties in a relatively short period of time by simulating the principles of natural evolution. This method has broad application potential in the fields of drug development, enzyme engineering, materials science, etc. According to the embodiments of the present invention, the "protein directed evolution system" herein is named "EvoScan system".

[0032] In this article, the term "regional" means that the EvoScan system is different from the traditional directed evolution system. It uses CRISPR-cas9-based gene targeted editing technology to apply the enCas9-PolIM5 complex used for diversity introduction in directed evolution to a more precise, limited-length region of the target gene, and introduces mutations into different regions of the entire gene for directed evolution by designing appropriate selection, screening, and iteration methods, thereby achieving the purpose of "regional" directed evolution.

[0033] In this article, the terms "CRISPR-Cas-DNA polymerase system", "ZFNs-DNA polymerase system", and "TALENs-DNA polymerase system" refer to a combination of a gene editing system and a polymerase system. According to an embodiment of the present invention, a "CRISPR-Cas-DNA polymerase system" refers to a composite system that has both the editing function of a "CRISPR-Cas system" and the polymerization repair function of a "DNA polymerase system". According to an embodiment of the present invention, a "CRISPR-Cas-DNA polymerase system" is an "enCas9-PolIM5 complex protein", in which the Cas9 nickase can be modified to perform single-stranded cutting of the replication form of the M13 genome with the help of a guide RNA (gRNA), and the protein polymerase I that loses the mismatch repair ability after mutation introduces new bases by repairing the nicks on the M13 genome. In this process, there is a certain probability of introducing mutations, and finally the nicks are repaired by the DNA ligase in the host. The repair function of polymerase I mainly covers the characteristics of about 20 bases targeted by gRNA, and mutants can be efficiently introduced in a relatively limited area.

[0034] In this article, the term "multi-region scanning mutation" is a strategy in protein directed evolution for exploring and optimizing multiple regions of a protein at the same time. In a protein structure, different regions may have an important impact on the function and properties of a protein, so by introducing mutations and evaluating multiple regions at the same time, the performance of a protein can be more comprehensively understood and changed. According to an embodiment of the present invention, in a multi-region scanning mutation, a technician in the field selects multiple protein regions that need to be concerned, usually based on existing structural and functional information. Then, in these selected regions, a protein variant library is created by introducing a variety of mutations (such as amino acid substitutions, insertions or deletions). Next, these protein variants will undergo a screening or selection process to select variants with target performance or function. The screening method can be determined according to specific application requirements and may involve enzyme activity determination, ligand binding experiments, high-throughput screening, etc. Through the method of multi-region scanning mutation, multiple protein regions can be optimized simultaneously in the same round of evolution, thereby more effectively changing the properties and functions of proteins. This strategy can help technicians in the field to more comprehensively explore and transform proteins in protein engineering to meet specific application requirements.

[0035] In this article, the term "adaptive landscape" refers to the concept of the relationship between a protein sequence and its adaptability (such as function, stability, etc.). It can be viewed as a multidimensional space in which each point represents a possible protein sequence, and the height (or energy) of each point represents the adaptability of the protein in the biological system. The adaptive landscape can help those skilled in the art understand the various properties and functions in the protein sequence space. In the adaptive landscape, neighboring points of a protein sequence represent variants with similar sequences, while farther points represent sequences with greater differences. Lower energy or height indicates higher adaptability, while higher energy or low degree indicates lower adaptability.

[0036] The present invention proposes a regional protein directed evolution system and a method for regional directed evolution of a target protein, which will be described in detail below.

[0037] Domain-specific protein directed evolution system

[0038] In one aspect of the present invention, the present invention proposes a regional protein directed evolution system. According to an embodiment of the present invention, the system includes: an element to be directed evolution, a gene-targeted mutation element, and a phage-directed screening element, wherein the element to be directed evolution is arranged on a phage genome vector, and the phage-directed screening element of the phage genome vector is missing; the phage-directed screening element is arranged on a second vector, and the second vector is suitable for expression in bacteria or fungi; the phage-directed screening element is arranged on a second vector, and the second vector is suitable for expression in bacteria or fungi; the gene-targeted mutation element includes a gene-directed evolution tool and a gene-targeted indicator element, and the gene-directed evolution tool and the gene-targeted indicator element are arranged on the same or different vectors, preferably on different vectors;

[0039] The expression product of the gene directed evolution tool is suitable for performing region-based directed evolution processing on the element to be directed evolution, and the gene directed evolution tool is arranged on the third vector, and the third vector is suitable for expression in bacteria or fungi; the gene targeting indicator element is arranged on the second vector; the expression product of the element to be directed evolution regulates the expression of the phage directed screening element, and the activity of the expression product of the element to be directed evolution is positively correlated with the expression of the phage directed screening element. By using the CRISPR-cas9 system based on gRNA targeting as a directed method for introducing mutations, the scope of mutation introduction will be limited by the length of the gRNA targeting sequence and the accessibility of DNA polymerase repair. By utilizing this feature, different mutations and mutation combinations can be introduced relatively independently in different regions of the sequence, and in this way, the effect of regional directed evolution can be achieved. According to the system of an embodiment of the present invention, it is named EvoScan system. The schematic diagram of the EvoScan system is shown in the figure. Figure 1As shown, the core of the EvoScan system involves two biological units: M13 phage as the expression vector of the protein to be evolved, and Escherichia coli S2060 as the host for phage mutation screening. The inventor inserted the coding sequence corresponding to the protein to be evolved into the genome of the M13 phage, replacing the original core gene III (gIII) that helps phage infection and release, and called it Selection Phage (SP). At the same time, the expression of gIII is placed in the host cell, and the expression of gIII is coupled with the function of the protein to be evolved by designing an accessory plasmid (Accessory Plasmid, AP), and a targeted mutation plasmid (Targetedmutagenesis Plasmid, TP) is designed to perform genomic mutations on the target gene to be evolved. SP will infect the S2060 host bacteria containing AP and TP, and its single-stranded DNA will be replicated into double-stranded and gene expression will be performed. In this process, TP can introduce targeted mutations to this double-stranded DNA. The design of TP uses the enCas9-PolIM5 complex protein in the EvolvR system of Halperin, SO et al. CRISPR-guided DNA polymerases enabled virification of all nucleotides in a tunable window. Nature 560, 248-252 (2018). The Cas9 nickase can be modified to perform single-stranded cutting on the replication form of the M13 genome with the help of guide RNA (gRNA), and the protein polymerase I that loses the mismatch repair ability after mutation introduces new bases by repairing the nicks on the M13 genome. There is a certain probability of introducing mutations in this process, and finally the nicks are repaired by the DNA ligase in the host. The repair function of polymerase I mainly covers the characteristics of about 20 bases targeted by gRNA, and mutants can be efficiently introduced in a relatively limited area. The target protein mutant expressed by the mutated gene will control the expression of gIII in the cell through the assistance of AP, thereby controlling the number of SPs released from S2060. Taking advantage of the fact that M13 infection blocks other individuals from continuing to infect the host, reasonable AP design can make the functional activity of the protein positively correlated with proliferation, that is, the more active the mutant, the more SP it can release. The released SP is collected, and the concentration will be diluted in the next host system after passage. Mutants with relatively high activity will be enriched during the passage process and continue to infect in the next passage; while individuals with low activity will be gradually eliminated in this process.This high-efficiency, multi-round directed evolution method has the advantages of simplicity, efficiency and low cost compared to traditional random sequence library construction, mismatch PCR library construction and other methods, as well as the directed evolution system of repeatedly constructing expression systems through non-continuous vectors. Because the screening system is highly coupled to the functional activity of the protein, most of the mutants obtained by screening have higher functional activity than the original protein. Compared with random library construction and computational function prediction, the proportion of functionally active mutants that can be obtained is relatively higher. The latter usually obtains most mutations that cause protein activity to be lost or even inactivated, and the efficiency of obtaining active mutants is relatively low and the cost is more expensive. Proteins vary greatly in size, and a gRNA can usually only control mutations of 7-8 amino acids more efficiently, so a large number of gRNAs that meet the PAM site design need to be designed. Therefore, the inventors chose to insert the gRNA expression frame on the AP to construct multiple APs to evolve different sites of the protein separately, such as. Figure 2 and Figure 3 As shown. By introducing mutations through evolution at different sites, the combination of mutation sites is achieved, and then multiple intermediate mutants that meet the functional activity of the protein are discovered. Compared with other phage continuous evolution systems, the active intermediate mutants captured by EvoScan will provide more comprehensive help for subsequent studies on the adaptive landscape of proteins.

[0040] According to an embodiment of the present invention, based on different gene circuit regulation, the protein directed evolution system includes a protein-protein directed evolution system, a protein-small molecule directed evolution system and a protein-nucleic acid directed evolution system.

[0041] According to an embodiment of the present invention, the second carrier includes a second carrier No. 1, a second carrier No. 2 and a second carrier No. 3.

[0042] Among them, it needs to be explained that according to the embodiments of the present invention, the second vector No. 1 in the present invention corresponds to the protein-protein directed evolution system, the second vector No. 2 corresponds to the protein-small molecule directed evolution system, and the second vector No. 3 corresponds to the protein-nucleic acid directed evolution system.

[0043] According to an embodiment of the present invention, the directed evolution process is performed in the following manner: using the gene directed evolution tool element to perform a cleavage process on the expression product of the element to be directed evolution, so as to introduce mutations into the cleavage product.

[0044] According to an embodiment of the present invention, the gene directed evolution tool element is selected from at least one of the CRISPR-Cas-DNA polymerase system, the ZFNs-DNA polymerase system and the TALENs-DNA polymerase system.

[0045] According to an embodiment of the present invention, the CRISPR-Cas protein in the CRISPR-Cas-DNA polymerase system includes at least one of Cas9 nickase (nCas9), enCas9, nSaCas9 and other homologous Cas9 nickases, modified Cas9 nickases, other Cas protein nickases that cut DNA double links, etc. It should be explained that Cas9 is a CRISPR-associated protein widely used in gene editing and genetic engineering. It is a nuclease in the CRISPR system that can form a complex with CRISPR RNA (crRNA) and the transcription unit RNA (tracrRNA) of the transcription unit to achieve genome editing by recognizing and cutting specific DNA sequences; enCas9 (enhanced Cas9) is a modified and optimized variant of Cas9, designed to improve its editing efficiency and specificity. enCas9 is usually achieved through structural modification, protein engineering or mutation. These improvements can include enhancing the binding ability of Cas9 to the target DNA sequence, improving the nuclease activity of Cas9 or reducing non-specific shearing; SaCas9 (Staphylococcus aureus Cas9) is another type of Cas9 protein from Staphylococcus aureus. Compared with traditional Cas9, SaCas9 has a smaller molecular size and is therefore more suitable for gene editing in cells in some cases. SaCas9 has potential advantages in some gene editing applications, especially when the size of the vector to be delivered into the cell is limited; enSaCas9 (enhanced SaCas9) is a modified and optimized variant of SaCas9, similar to the optimization of Cas9 by enCas9. enSaCas9 aims to improve the editing efficiency, specificity and other related properties of SaCas9. By improving and optimizing the structure and performance of SaCas9, enSaCas9 can better meet specific gene editing needs. Cas9, enCas9, SaCas9 and enSaCas9 are all CRISPR-associated proteins used for gene editing and engineering. Their improvement and optimization are aimed at improving editing efficiency, specificity and other related properties to expand the application scope and efficacy of CRISPR technology.

[0046] According to an embodiment of the present invention, the gene targeting indicator element includes a gRNA expression element, and the gRNA has a targeting binding activity of an expression product of the element to be directed evolution.

[0047] According to an embodiment of the present invention, the CRISPR-Cas protein in the CRISPR-Cas-DNA polymerase system is selected from at least one of Cas9 nickase, enCas9, nSaCas9 and modified Cas9 nickase.

[0048] According to an embodiment of the present invention, the DNA polymerase in the CRISPR-Cas-DNA polymerase system includes error-prone DNA polymerase I, preferably at least one of PolIM5 polymerase, PolIM3 polymerase and PolIM3-TBD polymerase.

[0049] According to an embodiment of the present invention, the CRISPR-Cas-DNA polymerase system is selected from at least one of Cas9-PolIM5 and enCas9-PolIM3-TBD. According to the system of an embodiment of the present invention, the protein to be evolved can be comprehensively and directed modified in a manner of high efficiency, low cost, and multiple capture of intermediate mutants. Among them, the Cas9-PolIM5 can efficiently modify the protein to be evolved.

[0050] According to an embodiment of the present invention, the second carrier and the third carrier are the same carrier or different carriers, preferably different carriers.

[0051] According to an embodiment of the present invention, the third vector further comprises: a first promoter element; and a first inducible element; wherein the first promoter element, the first inducible element, and the gene directed evolution tool element are operably connected.

[0052] According to an embodiment of the present invention, the third vector further includes a resistance gene element.

[0053] According to an embodiment of the present invention, the first promoter element is selected from at least one of a T7 promoter and a J23 promoter.

[0054] According to an embodiment of the present invention, the inducible element includes at least one of a pVanA-VanR inducible element, a pBAD-araC inducible element, a pTet-tetR element, and a pTac-LacI inducible element. According to an embodiment of the present invention, these inducible elements are sequences for regulating gene expression, and can induce the expression of a target gene through appropriate conditions or signals. Among them, the pVanA-VanR inducible element refers to the inducible element related to the pVanA-VanR system. The pVanA-VanR system is a broad-spectrum β-lactam antibiotic resistance system that can regulate the expression of resistance genes under specific conditions. The inducible element can achieve gene induction by introducing genes or sequences related to the pVanA-VanR system; the pBAD-araC inducible element refers to the inducible element related to the araC family. araC is a transcriptional regulatory protein that can bind to the ara operator sequence and regulate the expression of downstream genes. By introducing the araC inducible element or related genomic fragments, the induction of gene expression can be achieved; the pTet-tetR element refers to the Tet inducible element, which is a regulatory sequence from the Tn10 transposon. The Tet element can respond to the presence of tetracycline antibiotics and regulate the expression of downstream genes. The introduction of the Tet element or related gene fragments can achieve gene induction. In addition to the inducible elements mentioned above, the embodiments of the present invention may also use other inducible elements not mentioned, which are selected according to specific experimental requirements or specific conditions of the research object.

[0055] According to an embodiment of the present invention, the pVanA-VanR inducible element includes a VanR transcription factor and a pVanA promoter. According to an embodiment of the present invention, the VanR transcription factor is a protein associated with the pVanA-VanR system, which is involved in regulating the transcription of a specific gene. The VanR transcription factor can bind to a specific DNA sequence and regulate the expression of downstream genes by activating or inhibiting the transcription process. The pVanA promoter is a promoter sequence associated with the pVanA-VanR system, which is located in the upstream region of the target gene. The promoter is a key element of gene expression, which can provide binding sites for the transcription machinery and binding sites for regulatory proteins, thereby regulating the transcription initiation of the gene. By combining the VanR transcription factor and the pVanA promoter, the pVanA-VanR inducible element can achieve the induced expression of the target gene. The VanR transcription factor interacts with the pVanA promoter and binds to the pVanA promoter, thereby activating or inhibiting the transcription process of the target gene. It should be noted that the pVanA-VanR system and the pVanA promoter are specific examples of inducible elements in the present invention, which are used to illustrate the principle of gene regulation. In practical applications, other inducible elements or regulatory mechanisms may be used to achieve induced gene expression depending on specific needs and research purposes.

[0056] According to an embodiment of the present invention, the pBAD-araC inducible element includes araC transcription factor and pBAD promoter.

[0057] According to an embodiment of the present invention, the pTet-tetR inducible element includes a tetR transcription factor and a pTet promoter.

[0058] According to an embodiment of the present invention, the pTac-LacI inducible element includes LacI transcription factor and pTac promoter.

[0059] According to an embodiment of the present invention, the resistance gene element includes at least one selected from the group consisting of spectinomycin resistance gene, ampicillinase gene, kanamycin resistance gene, streptomycin resistance gene and chloramphenicol resistance gene. According to an embodiment of the present invention, these resistance genes refer to genes that can make host cells resistant to specific antibiotics or toxic compounds, which are usually derived from resistance genes in nature or engineered resistance genes, and can make host cells tolerant to corresponding antibiotics or toxic compounds under appropriate conditions.

[0060] According to an embodiment of the present invention, the third vector comprises, from the 5' end to the 3' end, a first promoter element, a VanR transcription factor, a pVanA promoter, a gene directed evolution tool element, and a resistance gene element.

[0061] According to an embodiment of the present invention, the third vector has a nucleotide sequence as shown in SEQ ID NO:1.

[0062] According to an embodiment of the present invention, the phage directed screening element includes gIII. According to an embodiment of the present invention, gIII is a gene in the bacteriophage genome, which encodes the release protein of phage particles. The phage directed screening element generally refers to the gene or sequence related to the phage release process, including the various components involved in the release of phage particles from host cells. After the phage infects the host cell, the phage genome can utilize the biosynthetic mechanism of the host cell to replicate and transcribe. At the right time, the phage needs to release the newly generated phage particles to infect other host cells. The protein encoded by the gIII gene in the phage directed screening element plays an important role in this release process. Specifically, the protein encoded by gIII may participate in the assembly and maturation process of phage particles, and interact with the host cell membrane, causing the rupture of the cell membrane, thereby releasing the phage particles.

[0063] According to an embodiment of the present invention, the following is a specific situation when the protein directed evolution system of the present invention is a protein-protein directed evolution system:

[0064] According to an embodiment of the present invention, the interaction between the expression product of the element to be directed evolution and the target regulates the generation of cIp22-cI434 tetramer and the expression of the phage directed screening element.

[0065] According to an embodiment of the present invention, the second vector further comprises a second inducible element.

[0066] According to an embodiment of the present invention, the second inducible element comprises a second promoter, a third promoter and a first repressor, the second promoter promotes the expression of the first repressor, and the first repressor is suitable for repressing the expression of the third promoter.

[0067] According to an embodiment of the present invention, the second inducing element is disposed on the second carrier.

[0068] According to an embodiment of the present invention, the second promoter is selected from at least one of T7 promoter and J23 series promoter, the third promoter is selected from at least one of pTac, pVanR, pTet and pBAD, and the first repressor is selected from at least one of LacI, VanR, tetR and araC.

[0069] According to an embodiment of the present invention, the first repressor is suitable for releasing the repression of the third promoter in an IPTG environment.

[0070] According to an embodiment of the present invention, the element to be directed evolved is connected to the cIp22 expression element, and the cIp22 is fused with the expression product of the element to be directed evolved and expressed; the cI434 expression element is connected to the element encoding the first target, and the cI434 expression element and the element encoding the first target are arranged on the second vector, and the cI434 is fused with the first target and expressed, and the expression product of the element to be directed evolved has the activity of binding to the first target.

[0071] According to an embodiment of the present invention, the first target is a receptor protein soluble in the recipient bacteria, and the expression product of the element to be directed to evolve includes a target evolved protein soluble in the recipient bacteria and interacting with the receptor protein, and the target evolved protein includes at least one of an antibody, a nanobody, a short peptide, a protein chaperone and a cascade transcription factor.

[0072] According to an embodiment of the present invention, the first target includes EGFP, and the expression product of the element to be directed evolution includes but is not limited to anti-EGFP nanoantibodies or other interacting proteins that can be normally expressed in a prokaryotic system.

[0073] According to an embodiment of the present invention, the cIp22-cI434 tetramer is suitable for inhibiting the expression of the fourth promoter by combining the first operator element, the fourth promoter is suitable for starting the expression of the second inhibitor, the second inhibitor is suitable for inhibiting the expression of the fifth promoter, the fifth promoter is suitable for starting the expression of the phage directional screening element, and the fourth promoter, the first operator element, the second inhibitor, the fifth promoter and the phage directional screening element are operably connected. According to an embodiment of the present invention, the inventors coupled protein interaction affinity with M13 phage gIII expression based on a reverse two-hybrid system (RTHS). Specifically, cI434 and cIp22 are two inhibitory elements that are highly homologous but do not interact with each other after transformation, and the tetramer combined can inhibit the two operator elements on the promoter p434, thereby inhibiting the promoter. The two interacting proteins can combine the two inhibitory elements of cI434 and cIp22 connected thereto, thereby achieving inhibition of the downstream promoter p434, and then positively correlate the interaction affinity with the expression of gIII by connecting the expression of an inhibitor PhlF. In addition, according to an embodiment of the present invention, this design utilizes the interaction and regulatory cascade effect between gene regulatory elements. The cIp22-cI434 tetramer can inhibit the expression of the fourth promoter by binding to the first operator element, thereby inhibiting the transcription and expression of the gene associated with the fourth promoter. The activation activity of the fourth promoter can be designed to regulate the expression of the second inhibitor. The expression of the second inhibitor inhibits the expression of the fifth promoter, thereby preventing the transcription and expression of the gene associated with the fifth promoter. Finally, the fifth promoter is designed to start the expression of the phage directional screening element, thereby regulating the release process of the phage. By connecting these gene regulatory elements together in a specific order, the cascade regulatory effect can be achieved, thereby achieving precise regulation of the target gene. It should be noted that the specific connection method and sequence design depend on the requirements of the regulatory elements used in the embodiment and the target gene. This design allows for precise regulation and modulation of gene expression to meet specific research objectives and application needs.

[0074] According to an embodiment of the present invention, the cI434 is fused with the first target expression element, the fourth promoter, the first operator element, the second repressor, and the fifth promoter on the No. 1 second vector.

[0075] According to an embodiment of the present invention, the fourth promoter includes p434. In an embodiment of the present invention, the fourth promoter uses p434 as its sequence to start the expression of the second repressor connected thereto. This means that the p434 sequence can induce the transcription and expression of the second repressor by interacting with appropriate transcription factors and regulatory proteins, thereby regulating the expression level of the gene associated with the second repressor.

[0076] According to an embodiment of the present invention, the fifth promoter is selected from at least one of pPhlF, pQacR, and pMcbR. In an embodiment of the present invention, the fifth promoter uses pPhlF as its sequence to initiate the expression of the phage directed screening element connected thereto. This means that the pPhlF sequence can induce the transcription and expression of the phage directed screening element by interacting with appropriate transcription factors and regulatory proteins, thereby regulating the release process of phage particles.

[0077] According to an embodiment of the present invention, the first operator element includes a p434 operator.

[0078] According to an embodiment of the present invention, the second inhibitor includes but is not limited to inhibitor proteins in the tetR family such as PhlF, QacR, Mcb, etc. that match the fifth promoter. In an embodiment of the present invention, the second inhibitor uses PhlF as its sequence to inhibit the expression of the fifth promoter connected thereto. This means that the PhlF sequence can inhibit the transcription and expression of the fifth promoter by interacting with appropriate transcription factors and regulatory proteins, thereby regulating the expression level of genes related to the fifth promoter.

[0079] According to an embodiment of the present invention, when the protein directed evolution system of the present invention is a protein-protein interaction directed evolution system, taking the evolution of EGFP nanoantibodies as an example, the E103K mutant is directed to evolve in different regions, and the nucleotide sequence of the gRNA used is shown in SEQ ID NO: 51 to SEQ ID NO: 54, corresponding to the CDR regions (CDR1-CDR3) of three E103K mutants, and an off-target gRNA, to perform four different evolutions on the E103K mutant in different regions. The same structure can be used to evolve at least one of antibodies, nanoantibodies, short peptides, protein chaperones, and cascade transcription factors.

[0080] According to an embodiment of the present invention, the following is a specific situation when the protein directed evolution system of the present invention is a protein-small molecule interaction directed evolution system:

[0081] According to an embodiment of the present invention, the cIp22 expression element is connected to the cI434 expression element, the cIp22 and the cI434 are expressed in fusion, and the expression product of the element to be directed evolution is suitable for cutting the fusion expression product of cIp22 and cI434.

[0082] According to an embodiment of the present invention, the cIp22 and the cI434 are connected via a connecting peptide.

[0083] According to an embodiment of the present invention, the connecting peptide has a recognition site for the expression product of the element to be directed evolution.

[0084] According to an embodiment of the present invention, the expression product of the element to be directed evolution comprises a protease that can be soluble expressed in the recipient bacteria, and the soluble expressed protease includes but is not limited to M pro , TEV protease and HCV protease.

[0085] According to an embodiment of the present invention, the cIp22-cI434 tetramer is suitable for inhibiting the expression of the sixth promoter by binding to the second operator element, and the sixth promoter is suitable for promoting the expression of the phage directed screening element, and the sixth promoter, the second operator element and the phage directed screening element are operably connected.

[0086] According to an embodiment of the present invention, the sixth promoter includes p434.

[0087] According to an embodiment of the present invention, the second operator element includes a p434 operator.

[0088] According to an embodiment of the present invention, the cIp22 and cI434 fusion expression element, the sixth promoter, and the second operator element are arranged on the second vector No. 2.

[0089] According to an embodiment of the present invention, when the protein directed evolution system of the present invention is a protein-small molecule interaction directed evolution system, the region-specific directed evolution M pro The nucleotide sequences of gRNA used are shown in SEQ ID NO:55 to SEQ ID NO:86 to target M pro Different regions evolve differently.

[0090] According to an embodiment of the present invention, the following is a specific situation when the protein directed evolution system of the present invention is a protein-nucleic acid interaction directed evolution system:

[0091] According to an embodiment of the present invention, the second vector No. 3 further includes an eighth promoter, a third repressor, and a seventh promoter, the expression product of the third repressor is suitable for inhibiting the expression of the phage directed screening element by inhibiting the activity of the seventh promoter, the expression product of the element to be directed evolution is suitable for inhibiting the expression of the eighth promoter, and the eighth promoter, the third repressor, and the seventh promoter are operably connected to the phage directed screening element.

[0092] According to an embodiment of the present invention, the expression product of the element to be directed-evolved inhibits the expression of the third inhibitor by inhibiting the expression activity of the eighth promoter.

[0093] According to an embodiment of the present invention, the third repressor is selected from tetR family transcription factors, such as at least one of PhlF, QacR and McbR.

[0094] According to an embodiment of the present invention, the expression product of the element to be directed evolution includes a protein that binds to a specific DNA sequence, preferably a transcription factor, more preferably a tetR family transcription factor, such as at least one of AmeR and McbR.

[0095] According to an embodiment of the present invention, the seventh promoter includes at least one of pPhlF, pQacR and pMcbR that matches the third repressor.

[0096] According to an embodiment of the present invention, the eighth promoter includes a DNA sequence that binds to the transcription factor to be evolved as a normally expressed promoter of the operator, preferably pAmeR.

[0097] According to an embodiment of the present invention, the seventh promoter is suitable for being orthogonal to the eighth promoter and its matching binding protein, and the eighth promoter is suitable for being orthogonal to the seventh promoter and its matching binding protein.

[0098] According to an embodiment of the present invention, the third repressor is PhlF, the expression product of the element to be directed evolution is AmeR, the seventh promoter is pPhlF, and the eighth promoter is pAmeR.

[0099] According to an embodiment of the present invention, the second vector further includes a ninth promoter, and the ninth promoter and the gRNA expression element are operably connected.

[0100] According to an embodiment of the present invention, the second vector No. 1 comprises, from the 5' end to the 3' end, the ninth promoter, the gRNA expression element, the third promoter, the cI434 and the first target fusion expression element, the fourth promoter, the first operator element, the second repressor, the fifth promoter, the phage directed screening element, the second promoter and the first repressor.

[0101] According to an embodiment of the present invention, the second vector No. 1 has a nucleotide sequence as shown in SEQ ID NO:2 to SEQ ID NO:5.

[0102] According to an embodiment of the present invention, the second vector No. 2 comprises, from the 5' end to the 3' end, the ninth promoter, the gRNA expression element, the third promoter, the cIp22 and cI434 fusion expression element, the sixth promoter, the second operator element, the phage directed screening element, the second promoter and the first repressor.

[0103] According to an embodiment of the present invention, the second vector No. 2 has a nucleotide sequence as shown in SEQ ID NO:6 to SEQ ID NO:37.

[0104] According to an embodiment of the present invention, the second vector No. 3 comprises, from the 5' end to the 3' end, the ninth promoter, the gRNA expression element, the eighth promoter, the third suppressor, the seventh promoter, and the phage directional screening element.

[0105] According to an embodiment of the present invention, the second vector No. 3 has a nucleotide sequence as shown in SEQ ID NO:38 to SEQ ID NO:50.

[0106] According to an embodiment of the present invention, the second vector is different from the third vector, and the second vector is selected from at least one of pUC, ColE1, p15A, CloDF13, pSC101 and pBR322; the third vector is selected from at least one of pUC, ColE1, p15A, CloDF13, pSC101 and pBR322.

[0107] According to an embodiment of the present invention, the second vector is a p15A vector. According to an embodiment of the present invention, p15A is a commonly used plasmid vector, which is widely used in molecular biology research and genetic engineering experiments, has a small size and a medium replication number, and is suitable for replication and expression in bacteria such as Escherichia coli.

[0108] According to an embodiment of the present invention, the third vector is a ColE1 vector. According to an embodiment of the present invention, ColE1 is a plasmid vector widely used in molecular biology research and genetic engineering experiments, which has a small size and a high replication number, and can be efficiently replicated and expressed in bacteria such as Escherichia coli.

[0109] According to an embodiment of the present invention, the bacteria or fungi include natural host bacteria, or non-natural host bacteria that have been genetically modified.

[0110] According to an embodiment of the present invention, the bacteria is Escherichia coli.

[0111] According to an embodiment of the present invention, the Escherichia coli is S2060.

[0112] According to an embodiment of the present invention, when the regional protein directed evolution system of the present invention is a protein-nucleic acid interaction directed evolution system, AmeR is directed to evolve in different regions, and the nucleotide sequence of the gRNA used is as shown in SEQ ID NO:87 to SEQ ID NO:99, to perform different evolutions on the AmeR regions.

[0113] A method for the modular directed evolution of target proteins

[0114] In the second aspect of the present invention, the present invention proposes a method for the block directed evolution of a target protein. According to an embodiment of the present invention, the method utilizes the protein directed evolution system of the first aspect of the present invention carrying the target protein to be directed evolution to transform the target protein to be directed evolution. According to the method of the embodiment of the present invention, the protein to be evolved can be comprehensively directed transformed in a manner of high efficiency, low cost, and multiple capture of intermediate mutants.

[0115] According to an embodiment of the present invention, the transformation process is achieved by: transducing the second vector and the third vector in the protein directed evolution system into the recipient bacteria; infecting the recipient bacteria with a phage carrying a phage directed screening element in the protein directed evolution system described in the first aspect of the present invention; and culturing the transduced and infected recipient bacteria so that the target protein to be directed evolved is transformed in the recipient bacteria. According to an embodiment of the present invention, during the culturing process, the target protein to be directed evolved will be transformed in the recipient bacteria, and specific culture conditions and selection pressures will be applied to promote the evolution and improvement of the target protein. According to the method of the embodiment of the present invention, the protein to be evolved can be comprehensively directed transformed in a manner of high efficiency, low cost, and multiple capture of intermediate mutants.

[0116] According to an embodiment of the present invention, the introduction of the second vector in the protein directed evolution system into the recipient bacteria is carried out in the presence of a first inducer.

[0117] According to an embodiment of the present invention, the introduction of the third vector in the protein directed evolution system into the recipient bacteria is carried out in the presence of a second inducer.

[0118] According to an embodiment of the present invention, the first inducer is selected from at least one of IPTG, vanillic acid and arabinose.

[0119] According to an embodiment of the present invention, the second inducer is selected from at least one of PTG, vanillic acid and arabinose.

[0120] According to an embodiment of the present invention, the first inducer is the same as or different from the second inducer, and preferably the first inducer is different from the second inducer.

[0121] According to an embodiment of the present invention, the concentration of IPTG is 0-1 mM, the concentration of vanillic acid is 0-100 μM, the concentration of tetracycline is 0-100 μM, and the concentration of arabinose is 0.5-2% (w / v).

[0122] According to an embodiment of the present invention, the culture treatment condition is culture in a shaking incubator for 12 to 18 hours.

[0123] According to an embodiment of the present invention, the temperature of the culturing treatment is 37°C.

[0124] According to an embodiment of the present invention, after the culture treatment, the product of the culture treatment is further centrifuged, and the centrifuged product is sequenced to obtain the nucleotide sequence of the modified target protein. According to an embodiment of the present invention, after the culture treatment, the product is centrifuged. Centrifugation is a common method for separating and precipitating suspended matter. Cells or cell fragments can be separated from the culture medium. The target protein or components related thereto can be separated by centrifugation; the product after the centrifugation is sequenced. Sequencing is a method for analyzing DNA or RNA sequences, which can determine the gene sequence of the protein. In an embodiment of the present invention, sequencing is used to determine the sequence of the modified target protein to verify whether the protein has undergone the expected modification. According to the method of an embodiment of the present invention, the evolved protein can be further subjected to comprehensive directional modification in a highly efficient, low-cost, and multi-captured intermediate mutant manner.

[0125] The sequence of the present invention is shown in the following table

[0126] Sequence Listing

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] The scheme of the present invention will be explained below in conjunction with embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product instructions are used. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0244] Embodiment 1:

[0245] This embodiment constructs Figure 4 The EvoScan system shown performs directed evolution of EGFP nanobody mutants.

[0246] The specific experimental process is as follows:

[0247] (1) Constructing the phage genome vector, the second vector No. 1, and the third vector.

[0248] Construction of phage genome vector (SP plasmid for short): Based on the existing M13 phage genome backbone, a ribosome binding site (RBS) of appropriate strength and a nanobody linked to cIp22 that interacts with EGFP were inserted after the gIII promoter. Subsequently, the glutamic acid at position 103 on the complementarity determining region 3 (CDR3) of the EGFP nanobody was mutated to lysine to destroy its interaction with EGFP; Construction of the second vector No. 1 (the second vector is referred to as AP plasmid, and the vector of the AP plasmid is p15A): The inventors first designed 4 different gRNAs (nucleotide sequences such as SEQ ID NO: 51 to SEQ ID NO: NO:54) corresponding to the CDR regions (CDR1-CDR3) of three E103K mutants, and an off-target gRNA, to perform four different evolutions of the E103K mutant, and connect the fluorescent protein EGFP to cI434, and construct it into four No. 1 second vectors. The AP plasmids are p15A promoter, gRNA1-4, pTac promoter, cI434-EGFP fusion expression product, p434 promoter, p434 operator, PhlF repressor, pPhlF promoter, gIII release element, p15A promoter, lacI repressor from 5' to 3' end, respectively. The nucleotide sequences of the four No. 1 second vectors are as shown in SEQ ID NO:2-SEQ ID NO:5 is shown; the third vector (TP plasmid for short, the vector of TP plasmid is CoIE1) is constructed: spectinomycin resistance is inserted into the plasmid at the ColE1 replication start site, and the pVanA-VanR system is added to induce the expression of the enCas9-PolIM5 protein complex with a certain toxicity, ensuring that it can minimize the toxic effect on the host cells when not infected by phages, and at the same time can efficiently express the mutant system when the evolution system is working.

[0249] (2) Construct receptor bacteria and carrier phage.

[0250] For the recipient bacteria, the inventors used the Inoue method to prepare S2060 super chemical competent cells, and transformed the above-constructed AP and TP plasmids into them respectively to obtain different recipient bacteria S2060-AP, TP.

[0251] For the vector phage, the inventors first obtained double-stranded fragments from the phage genome backbone by PCR, and then connected the RBS and the EGFP nanobody E103K mutant to form a complete circular double-stranded DNA.

[0252] Prepare S2208 (S2060 + pJC175e) chemical competent cells and transform the constructed fragments into them. There is a promoter activated by phage infection on pJC175e, which can start the expression of downstream gIII. The other proteins of the phage are provided by the transformed skeleton, so that new phages can be produced without gIII on the phage genome. The phages will be collected by centrifugation to collect the supernatant, and the S2208 cells in the supernatant will be filtered out through a 0.22 micron bacterial filter.

[0253] (3) Search for areas with evolutionary potential.

[0254] For each type of S2060-AP and TP, the inventors performed discontinuous evolution of phages. One generation of phages followed the following steps: First, the saturated recipient bacteria cultured overnight were transferred to the rapid proliferation phase (log phase) and a concentration of 5×10 7 pfu / mL of SP, and 50μM vanillic acid to induce the expression of the protein complex responsible for mutation on TP, thereby causing the added SP to mutate. 1mM IPTG was added to control the expression of the target protein and then the expression of gIII to help the proliferation of M13 phage. The entire system was cultured overnight in a shaker at 37°C for no less than 16 hours. The next day, the system was centrifuged and the supernatant was taken as the next batch of phage for passage. For the obtained phage, the sequence fragment of the gene to be evolved was obtained by PCR. The concentration of the existing phage system can be determined by plaque assay, and the sequencing results of the E103K mutant of the EGFP nanoantibody evolved by the EvoScan system were obtained by Sanger sequencing.

[0255] The results are as follows Figure 5 As shown, after two generations of passage, only the group gRNA3 corresponding to CDR3 had an increase in phage titer, while the other three groups continued to decline until the fourth passage. After sequencing the four groups of phages in this example, it can be found that the group targeted by gRNA3 has mutated, and the lysine at position 103 has returned to glutamic acid, which shows that the EvoScan system constructed in this example has the ability to orientate the evolution of the EGFP nanobody E103K mutant.

[0256] Embodiment 2:

[0257] In this embodiment, the method in Example 1 is used to construct Figure 6 The EvoScan system shown is for M pro Perform systematic mutation. The experimental process different from Example 1 is as follows:

[0258] Construction of phage genome vector and second vector No. 2

[0259] Construction of phage genome vector (SP plasmid): Based on the existing M13 phage genome backbone, a ribosome binding site (RBS) with appropriate strength and M pro ; Construction of the second vector No. 2 (the second vector is referred to as AP plasmid, and the vector of AP plasmid is p15A): The inventors designed 32 different gRNAs (nucleotide sequences are shown in SEQ ID NO: 55 to SEQ ID NO: 86) to cover the entire M pro gene, construct 32 No. 2 second vectors, the AP plasmids are respectively p15A promoter, gRNA5-36, pTac promoter, cIp22-cI434 fusion expression product, p434 promoter, p434 operator, gIII release element, p15A promoter, lacI repressor from 5' to 3' end, and the nucleotide sequences of the 32 No. 2 second vectors are shown in SEQ ID NO:6 to SEQ ID NO:37, respectively;

[0260] After eight passages, the inventors sequenced the obtained phage. Figure 7 As shown, the generated mutations are distributed throughout the gene, and some mutations such as F140L, E166V, S144A have been disclosed, which to a certain extent proves the reliability of the system constructed in this embodiment. Among them, many mutation sites have not been disclosed, such as S62, L75, N119, S144, T169, A191, P241, and G302, etc., indicating that the EvoScan system can evolve new mutants more comprehensively.

[0261] Embodiment 3:

[0262] In this embodiment, the method in Example 1 is used to construct Figure 8 The EvoScan system shown in the figure performs directed evolution on AmeR. The experimental process different from that in Example 1 is as follows:

[0263] Construction of phage genome vector and second vector No. 3

[0264] Construction of phage genome vector (SP plasmid for short): Based on the existing M13 phage genome backbone, a ribosome binding site (RBS) and AmeR of appropriate strength were inserted after the gIII promoter; Construction of the second vector No. 3 (the second vector is referred to as AP plasmid, and the vector of the AP plasmid is p15A): The inventors designed 13 different gRNAs (SEQ ID NO: 87 to SEQ ID NO: 99) to cover the entire AmeR gene, and constructed 13 second vectors No. 2. The AP plasmids are p15A promoter, gRNA37 to 49, pAmeR promoter, PhlF repressor, pPhlF promoter, and gIII release element from the 5' end to the 3' end, respectively. The nucleotide sequences of the 13 second vectors No. 3 are shown in SEQ ID NO: 38 to SEQ ID NO: 50, respectively;

[0265] The results are as follows Figure 7 As shown in FIG. 1 , after four passages, 8 groups were finally enriched with effective mutations in the supernatant. Using these 8 groups, the inventors randomly designed the following Fig. 9 The inventors followed the 8 evolutionary paths shown, and collected mutants at each step of each path. Ultimately, they collected a total of 82 mutants, involving 52 mutations and 39 amino acid residues.

[0266] For the above mutants, the inventors designed Fig.10 The inhibitory effect of mutants was tested using the fluorescence detection system shown. Fig.10 As shown in the figure, it can be seen that most mutants have improved inhibitory effects compared to the wild type, and some mutants may have improved effects by more than 20 times, which shows the effectiveness of the EvoScan system.

[0267] Embodiment 4:

[0268] This embodiment is basically the same as embodiment 2, except that the method in embodiment 2 is used to construct Fig.15 The reverse two-hybrid system shown in the figure systematically mutates HCV protease and detects its cleavage activity. Among them, the experimental process different from Example 2 is as follows: HCV protease is selected in the reverse two-hybrid system for flow fluorescence test to verify the cleavage activity. The specific construction is: vanillic acid controls the induction expression of protease, and IPTG controls the expression of cI434-protease substrate-cIp22 complex. The specific experimental concentrations are: IPTG: 100μM, vanillic acid: 0, 50, 100, 200μM. The test results are shown in Fig.16 As shown, as the protein expression level increases, the fluorescence rises after cleavage, indicating that it can reflect the protease cleavage activity.

[0269] Comparative Example 1:

[0270] This comparative example is constructed by the method in reference Miller SM, Wang T, Liu DR. Phage-assisted continuous and non-continuous evolution. Nat Protoc. 2020 Dec; 15(12): 4101-4127. Fig.11 The phage-assisted non-continuous evolution (PANCE) system shown in the embodiment 1 is used to direct the evolution of EGFP nanoantibodies. Compared with the EvoScan system in Example 1, the difference between the two systems is that the targeted TP and non-targeted MP6 mutant plasmids are different. Specifically, the partial function of Cas9 in the TP plasmid core expression complex protein enCas9-PolIM5 requires gRNA to guide the targeting to a specific area, and then introduce mutations through a DNA polymerase with a high mismatch rate; while the MP6 mutant plasmid system does not have this CRISPR-cas system, and the way it introduces mutations is to introduce direct base mutations by deamination and the like, and to affect the accuracy of mismatch repair in the evolutionary system (the specific structure of MP6 is shown in FIG. Fig.14 As shown), therefore, gRNA does not need to be inserted into the AP plasmid in this comparative example. The AP plasmid in this comparative example is composed of pTac promoter, cI434-EGFP fusion expression product, p434 promoter, p434 operator, PhlF repressor, pPhlF promoter, gIII release element, p15A promoter, and lacI repressor from 5' to 3' end, and the EGFPE103K mutant was directed evolved using the PANCE system.

[0271] The results are as follows Fig.11 As shown, after 8 PANCE passages and sequencing of the passaged products, the inventors found that no new mutations were introduced into the nanobody, but after five generations, an N29D mutation appeared on cIp22, which was verified to cause the two cI inhibitory elements to self-bind without the need for the proteins they connected to interact. This shows that for the target site, the TP of the protein directed evolution system (EvoScan system) constructed in Example 1 is more efficient than the MP6 mutation of the PANCE system constructed in this comparative example 1.

[0272] Comparative Example 2:

[0273] Similar to Comparative Example 1, this comparative example uses the method in Comparative Example 1 to construct a PANCE system for M proCompared with the EvoScan system in Example 2, the difference between the two systems is that the targeted TP and non-targeted MP6 mutation plasmids are different. Therefore, gRNA does not need to be inserted into the AP plasmid in this comparative example. Referring to the method in Comparative Example 1, the PANCE system is used to mutate M pro The mutants were subjected to directed evolution. The experimental process different from that of comparative example 1 is as follows: the AP plasmids are respectively pTac promoter, cIp22-cI434 fusion expression product, p434 promoter, p434 operator, gIII phage directed screening element, p15A promoter, and lacI repressor from 5' to 3' end.

[0274] The results are as follows Fig.12 As shown, the PANCE system is designed for M pro Systematic mutagenesis still failed to enrich effective mutations after 36 passages, but after 96 passages, the four groups were enriched with mutants with higher escape effects, including N119D and A191V obtained by the EvoScan system in Example 2 after only 8 passages. These results prove that the EvoScan system constructed in Example 2 is more efficient than the PANCE system constructed in Comparative Example 2.

[0275] Comparative Example 3:

[0276] Similar to Comparative Example 1, this comparative example is constructed by the method in Comparative Example 1. Fig.13 The PANCE system shown is directed evolution for AmeR. Compared with the EvoScan system in Example 3, the difference between the two systems is that the targeted TP is different from the non-targeted MP6 mutant plasmid, so there is no need to insert gRNA into the AP plasmid in this comparative example. Referring to the method in comparative example 1, the PANCE system is used to direct evolution of AmeR. Among them, the experimental process different from comparative example 1 is as follows: the AP plasmid is respectively pAmeR promoter, PhlF repressor, pPhlF promoter, and gIII release element from the 5' end to the 3' end.

[0277] The results are as follows Fig.13 As shown, after multiple passages, the PANCE system only obtained two mutations, S57R and R43S. Compared with the EvoScan system in Example 3, which obtained 8 significant mutations after 4 passages, the evolutionary efficiency of the PANCE system constructed in this comparative example is lower.

[0278] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0279] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention, and that those skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A domain-specific protein directed evolution system, characterized in that: include: Elements to be directed evolution, gene targeted mutation elements and phage directed screening elements, Wherein, the element to be directed evolution is set on a phage genome vector, and the phage directed screening element of the phage genome vector is missing; The phage directed selection element is disposed on a second vector, and the second vector is suitable for expression in bacteria or fungi; The gene targeting mutation element comprises a gene directed evolution tool and a gene targeting indicator element, and the gene directed evolution tool and the gene targeting indicator element are arranged on the same or different vectors, preferably on different vectors; The expression product of the gene directed evolution tool is suitable for performing region-based directed evolution processing on the element to be directed evolution, and the gene directed evolution tool is arranged on a third vector, and the third vector is suitable for expression in bacteria or fungi; The gene targeting indicator element is disposed on the second vector; The expression product of the element to be directed evolution regulates the expression of the phage directed screening element, and the activity of the expression product of the element to be directed evolution is positively correlated with the expression of the phage directed screening element; Optionally, the sub-regional protein directed evolution system includes a protein-protein directed evolution system, a protein-small molecule directed evolution system, and a protein-nucleic acid directed evolution system; Optionally, the second carrier includes second carrier No. 1, second carrier No. 2 and second carrier No.

3.

2. The regional protein directed evolution system according to claim 1, characterized in that: The directed evolution process is performed in the following manner: using the gene directed evolution tool to perform a cleavage process on a specific region of the gene of the element to be directed evolution, so as to introduce a mutation into the cleavage product; Optionally, the gene directed evolution tool element is selected from at least one of a CRISPR-Cas-DNA polymerase system, a ZFNs-DNA polymerase system, and a TALENs-DNA polymerase system; Optionally, the gene targeting indicator element comprises a gRNA expression element, wherein the gRNA has a targeting binding activity to an expression product of the element to be directed evolution; Optionally, the CRISPR-Cas protein in the CRISPR-Cas-DNA polymerase system is selected from at least one of Cas9 nickase, enCas9, nSaCas9 and modified Cas9 nickase; Optionally, the DNA polymerase in the CRISPR-Cas-DNA polymerase system includes error-prone DNA polymerase I, preferably at least one of PolIM5 polymerase, PolIM3 polymerase and PolIM3-TBD polymerase; Optionally, the CRISPR-Cas-DNA polymerase system is selected from at least one of enCas9-PolIM5 and enCas9-PolIM3-TBD.

3. The regional protein directed evolution system according to claim 2, characterized in that: The second carrier and the third carrier are the same carrier or different carriers, preferably different carriers.

4. The regional protein directed evolution system according to claim 2 or 3, characterized in that: The third carrier further comprises: a first promoter element; and first inducing element; Wherein, the first promoter element, the first inducible element, and the gene directed evolution tool element are operably connected; Optionally, the third vector further comprises a resistance gene element; Optionally, the first promoter element is selected from at least one of a T7 promoter and a J23 promoter; Optionally, the first inducible element is selected from at least one of a pVanA-VanR inducible element, a pBAD-araC inducible element, a pTet-tetR inducible element, and a pTac-LacI inducible element; Optionally, the pVanA-VanR inducible element includes a VanR transcription factor and a pVanA promoter; Optionally, the pBAD-araC inducible element comprises an araC transcription factor and a pBAD promoter; Optionally, the pTet-tetR inducible element comprises a tetR transcription factor and a pTet promoter; Optionally, the pTac-LacI inducible element includes a LacI transcription factor and a pTac promoter; Optionally, the resistance gene element is selected from at least one of a spectinomycin resistance gene, an ampicillinase gene, a kanamycin resistance gene, a streptomycin resistance gene and a chloramphenicol resistance gene; Optionally, the third vector comprises, from the 5' end to the 3' end, a first promoter element, a transcription factor, a promoter regulated by a transcription factor, a gene directed evolution tool element, and a resistance gene element; Optionally, the third vector has the nucleotide sequence shown in SEQ ID NO:

1.

5. The domain-specific protein directed evolution system according to claim 1, characterized in that: The phage directed selection element includes gIII.

6. The domain-specific protein directed evolution system according to claim 1, characterized in that: The interaction between the expression product of the element to be directed evolution and the target regulates the generation of protein-protein interaction polymers to regulate the expression of the phage directed screening element; Optionally, the second vector further comprises a second inducible element; Optionally, the second inducible element comprises a second promoter, a third promoter and a first repressor, the second promoter promotes the expression of the first repressor, and the first repressor is suitable for repressing the expression of the third promoter; Optionally, the second inducible element is disposed on the second vector; Optionally, the second promoter is selected from at least one of a T7 promoter and a J23 promoter, the third promoter is selected from at least one of pTac, pVanR, pTet and pBAD, and the first repressor is selected from at least one of LacI, VanR, tetR and araC; Optionally, the first repressor is suitable for derepressing the third promoter in the context of a corresponding inducer.

7. The domain-specific protein directed evolution system according to claim 6, characterized in that: The element to be directed evolution is connected to the cIp22 expression element, and the cIp22 is fused with the expression product of the element to be directed evolution; The cI434 expression element is connected to the element encoding the first target, the cI434 expression element and the element encoding the first target are arranged on the second vector, the cI434 is fused with the first target and expressed, and the expression product of the element to be directed evolution has the activity of binding to the first target; Optionally, the first target is a receptor protein soluble in the recipient bacteria, the expression product of the element to be directed evolution includes a target evolution protein soluble in the recipient bacteria and interacting with the receptor protein, the target evolution protein includes at least one of an antibody, a nanobody, a short peptide, a protein chaperone and a cascade transcription factor; Optionally, the first target includes EGFP, and the expression product of the element to be directed evolution includes an anti-EGFP nanobody.

8. The domain-specific protein directed evolution system according to claim 6, characterized in that: The cIp22-cI434 tetramer is suitable for inhibiting the expression of a fourth promoter by binding to a first operator element, the fourth promoter is suitable for initiating the expression of a second inhibitor, the second inhibitor is suitable for inhibiting the expression of a fifth promoter, the fifth promoter is suitable for initiating the expression of the phage directed selection element, and the fourth promoter, the first operator element, the second inhibitor, the fifth promoter and the phage directed selection element are operably connected; Optionally, the cI434 is fused with the first target expression element, the fourth promoter, the first operator element, the second repressor, and the fifth promoter on the second vector No. 1; Optionally, the fourth promoter comprises p434; Optionally, the fifth promoter is selected from at least one of pPhlF, pQacR and pMcbR; Optionally, the first operator element comprises a p434 operator; Optionally, the second inhibitor is selected from at least one of Ph1F, QacR and McbR.

9. The domain-specific protein directed evolution system according to claim 6, characterized in that: The cIp22 expression element is connected to the cI434 expression element, the cIp22 and the cI434 are fused and expressed, and the expression product of the element to be directed evolution is suitable for cutting the fusion expression product of cIp22 and cI434 under the action of small molecules; Optionally, the cIp22 and the cI434 are connected via a connecting peptide; Optionally, the connecting peptide has a recognition site for the expression product of the element to be directed evolution; Optionally, the expression product of the element to be directed evolution comprises a protease that can be soluble and expressed in a recipient bacterium, preferably one of Mpro, TEV protease and HCV protease.

10. The region-specific protein directed evolution system according to claim 9, characterized in that: The cIp22-cI434 tetramer is suitable for inhibiting the expression of a sixth promoter by binding to a second operator element, the sixth promoter is suitable for promoting the expression of the bacteriophage directed selection element, and the sixth promoter, the second operator element and the bacteriophage directed selection element are operably connected; Optionally, the sixth promoter comprises p434; Optionally, the second operator element comprises a p434 operator; Optionally, the cIp22 and cI434 fusion expression element, the sixth promoter, and the second operator element are set on the second vector No.

2.

11. The domain-specific protein directed evolution system according to claim 1, characterized in that: The second vector No. 3 further comprises an eighth promoter, a third repressor, and a seventh promoter, the expression product of the third repressor is suitable for inhibiting the expression of the phage directed screening element by inhibiting the activity of the seventh promoter, the expression product of the element to be directed evolution is suitable for inhibiting the expression of the eighth promoter, and the eighth promoter, the third repressor, the seventh promoter and the phage directed screening element are operably connected; Optionally, the expression product of the element to be directed-evolved inhibits the expression of the third inhibitor by inhibiting the expression activity of the eighth promoter; Optionally, the third inhibitor is selected from at least one of PhlF, QacR and McbR; Optionally, the expression product of the element to be directed evolution includes a protein that binds to a specific DNA sequence, preferably a transcription factor, more preferably at least one of AmeR and McbR; Optionally, the seventh promoter includes at least one of pPhlF, pQacR and pMcbR that matches the third repressor; Optionally, the eighth promoter includes a DNA sequence that binds to the transcription factor to be evolved as a normally expressed promoter of the operator, preferably pAmeR; Optionally, the seventh promoter is adapted to be orthogonal to the eighth promoter and its cognate binding protein, and the eighth promoter is adapted to be orthogonal to the seventh promoter and its cognate binding protein.

12. The domain-specific protein directed evolution system according to claim 2, characterized in that: The second vector further includes a ninth promoter, and the ninth promoter and the gRNA expression element are operably connected.

13. The domain-specific protein directed evolution system according to any one of claims 6 to 8 and 12, characterized in that: The second vector No. 1 is composed of the ninth promoter, the gRNA expression element, the third promoter, the cI434 and the first target fusion expression element, the fourth promoter, the first operator element, the second repressor, the fifth promoter, the phage directional screening element, the second promoter and the first repressor from the 5' end to the 3' end; Optionally, the second vector No. 1 has a nucleotide sequence as shown in SEQ ID NO:2 to SEQ ID NO:

5.

14. The domain-specific protein directed evolution system according to any one of claims 6, 9 to 10, and 12, characterized in that: The second vector No. 2 has, from the 5' end to the 3' end, the ninth promoter, the gRNA expression element, the third promoter, the cIp22 and cI434 fusion expression element, the sixth promoter, the second operator element, the phage directional screening element, the second promoter and the first repressor; Optionally, the second vector No. 2 has a nucleotide sequence as shown in SEQ ID NO:6 to SEQ ID NO:

37.

15. The domain-specific protein directed evolution system according to claim 11 or 12, characterized in that: The second vector No. 3 has the ninth promoter, the gRNA expression element, the eighth promoter, the third suppressor, the seventh promoter, and the phage directional screening element from the 5' end to the 3' end; Optionally, the second vector No. 3 has a nucleotide sequence as shown in SEQ ID NO: 38 to SEQ ID NO:

50.

16. The protein directed evolution system according to claim 1, characterized in that: The second vector is different from the third vector, the second vector is selected from at least one of pUC, ColE1, p15A, CloDF13, pSC101 and pBR322; the third vector is selected from at least one of pUC, ColE1, p15A, CloDF13, pSC101 and pBR322.

17. The domain-specific protein directed evolution system according to claim 1 or 2, characterized in that: The bacteria or fungi include natural host bacteria, or non-natural host bacteria that have been genetically modified; Optionally, the bacteria is Escherichia coli; Optionally, the Escherichia coli is S2060.

18. A method for directed evolution of a target protein in a modular manner, characterized in that: The target protein to be directed evolution is transformed using the protein directed evolution system according to any one of claims 1 to 17 carrying the target protein to be directed evolution.

19. The method according to claim 18, characterized in that The transformation process is achieved by: Transducing the second vector and the third vector in the protein directed evolution system into a recipient bacterium; Infecting the recipient bacteria with a phage carrying a phage directed screening element in the protein directed evolution system according to any one of claims 1 to 17; as well as The transduced and infected recipient bacteria are cultured so that the target protein to be directed-evolved can be transformed in the recipient bacteria.

20. The method according to claim 19, characterized in that The introduction of the second vector in the protein directed evolution system into the recipient bacteria is carried out under the condition of the presence of the first inducer; Optionally, the introduction of the third vector in the protein directed evolution system into the recipient bacteria is carried out in the presence of a second inducer; Optionally, the first inducing agent is selected from at least one of IPTG, vanillic acid, tetracycline and arabinose; Optionally, the second inducing agent is selected from at least one of IPTG, vanillic acid, tetracycline and arabinose; Optionally, the first inducer is the same as or different from the second inducer, preferably the first inducer is different from the second inducer; Optionally, the concentration of IPTG is 0-1 mM, the concentration of vanillic acid is 0-100 μM, the concentration of tetracycline is 0-100 μM, and the concentration of arabinose is 0.5-2% (w / v); Optionally, the culture treatment condition is cultured in a shaker for 12 to 18 hours; Optionally, the temperature of the culture treatment is 37°C; Optionally, after the culture treatment, the method further comprises centrifuging the culture treatment product and sequencing the centrifuged product to obtain the nucleotide sequence of the modified target protein.

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