Tev protease mutants and uses thereof
By introducing specific amino acid residue mutations into the TEV protease and optimizing its expression system, the problems of low expression levels and self-cleavage of the TEV protease in E. coli were solved, achieving efficient soluble expression and cost savings.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BLOOMATURE BIOTECHNOLOGY CO LTD
- Filing Date
- 2024-12-30
- Publication Date
- 2026-06-30
AI Technical Summary
Wild-type TEV protease is expressed at low levels in E. coli prokaryotic expression systems and is prone to intramolecular self-cleavage, resulting in high enzyme digestion efficiency and high costs for large-scale production.
By mutating specific amino acid residue sites of the TEV protease, mutant TEV proteases were constructed, including amino acid residue mutations at positions 230, 40, 60, 176, and 217, and their expression systems were optimized to improve expression levels and solubility.
This improved the expression level and solubility of TEV protease, reduced downstream purification costs, and provided a theoretical basis for large-scale production.
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Figure CN122303201A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of synthetic biology, and in particular to TEV protease mutants and their applications. Background Technology
[0002] TEV protease is a highly specific cysteine protease derived from Tobacco Etch Virus. It recognizes and cleaves a specific amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly / Ser, with the cleavage site between glutamine (Gln) and glycine / serine (Gly / Ser). Its molecular weight is approximately 27 kDa, and its gene sequence encodes 242 amino acids. Due to its high specificity and relatively mild reaction conditions, TEV protease has wide applications in the biotechnology field, such as removing affinity tags from purified recombinant fusion proteins to obtain purified target proteins, and performing precise protein processing in protein research and biopharmaceutical production.
[0003] Wild-type TEV protein exhibits low expression levels in E. coli prokaryotic expression systems, with most target proteins existing in the form of inclusion bodies and prone to intramolecular self-cleavage, significantly reducing the activity and cleavage efficiency of TEV protease. While genetic engineering can improve TEV protein expression to some extent, making the cost of small-scale laboratory preparation of TEV protease relatively low, large-scale production still requires large-scale fermentation and purification, resulting in high production costs. Therefore, there is still room for improvement in TEV protein expression levels through genetic engineering and protein engineering. Summary of the Invention
[0004] The purpose of this invention is to provide a TEV protease mutant to improve the expression level of TEV protease.
[0005] On one hand, this application provides a TEV protease mutant, the amino acid sequence of which comprises a sequence obtained by mutating an amino acid residue site of a wild-type TEV protease, wherein the amino acid residue site is selected from one or more of positions 230, 40, 60, 138, 176, and 217, and the wild-type TEV protease comprises the amino acid sequence shown in SEQ ID NO.1.
[0006] Preferably, the nucleic acid molecule encoding the wild-type TEV protease comprises a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.2.
[0007] Furthermore, the amino acid residue site mutation includes at least one or more of the following A1)-A6):
[0008] A1) The 230th amino acid residue is mutated from a glutamic acid residue (E) to a leucine residue (L);
[0009] A2) The 40th amino acid residue is mutated from a phenylalanine residue (F) to a tyrosine residue (Y);
[0010] A3) The 60th amino acid residue is mutated from a leucine residue (L) to a histidine residue (H);
[0011] A4) The 60th amino acid residue is mutated from a leucine residue (L) to a tryptophan residue (W);
[0012] A5) The 138th amino acid residue is mutated from isoleucine residue (I) to threonine residue (T);
[0013] A6) The 176th amino acid residue is mutated from an asparagine residue (N) to a valine residue (V);
[0014] The 217th amino acid residue in A7 is mutated from a phenylalanine residue (F) to a glutamine residue (Q).
[0015] Preferably, the TEV protease mutant is obtained by mutating the 40th amino acid residue of the wild-type TEV protease by changing a phenylalanine residue (F) to a tyrosine residue (Y); preferably, the TEV protease mutant comprises the amino acid sequence shown in SEQ ID NO. 5; preferably, the nucleic acid molecule encoding the protease mutant comprises the nucleotide sequence shown in SEQ ID NO. 6 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 6.
[0016] Preferably, the TEV protease mutant is obtained by mutating the 60th amino acid residue of the wild-type TEV protease by replacing a leucine residue (L) with a histidine residue (H); preferably, the TEV protease mutant comprises the amino acid sequence shown in SEQ ID NO. 7; preferably, the nucleic acid molecule encoding the protease mutant comprises the nucleotide sequence shown in SEQ ID NO. 8 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 8.
[0017] Preferably, the TEV protease mutant is obtained by mutating the 60th amino acid residue of the wild-type TEV protease by replacing a leucine residue (L) with a tryptophan residue (W); preferably, the TEV protease mutant comprises the amino acid sequence shown in SEQ ID NO. 9; preferably, the nucleic acid molecule encoding the protease mutant comprises the nucleotide sequence shown in SEQ ID NO. 10 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 10.
[0018] Preferably, the TEV protease mutant is obtained by mutating the 138th amino acid residue of the wild-type TEV protease from an isoleucine residue (I) to a threonine residue (T); preferably, the TEV protease mutant comprises the amino acid sequence shown in SEQ ID NO. 11; preferably, the nucleic acid molecule encoding the protease mutant comprises the nucleotide sequence shown in SEQ ID NO. 12 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 12.
[0019] Preferably, the TEV protease mutant is obtained by mutating the 176th amino acid residue of the wild-type TEV protease from an asparagine residue (N) to a valine residue (V); preferably, the TEV protease mutant comprises the amino acid sequence shown in SEQ ID NO. 13; preferably, the nucleic acid molecule encoding the protease mutant comprises the nucleotide sequence shown in SEQ ID NO. 14 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 14.
[0020] Preferably, the TEV protease mutant is obtained by mutating the 217th amino acid residue of the wild-type TEV protease from a phenylalanine residue (F) to a glutamine residue (Q); preferably, the TEV protease mutant comprises the amino acid sequence shown in SEQ ID NO. 15; preferably, the nucleic acid molecule encoding the protease mutant comprises the nucleotide sequence shown in SEQ ID NO. 16 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 16.
[0021] Further, the TEV protease mutant is obtained by mutating the glutamic acid residue at position 230 of the wild-type TEV protease to a leucine residue; preferably, the TEV protease mutant comprises the amino acid sequence shown in SEQ ID NO.17.
[0022] More preferably, the nucleic acid molecule encoding the TEV protease mutant comprises a nucleotide sequence as shown in SEQ ID NO. 6 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 18.
[0023] Furthermore, the amino acid residue site mutation also includes at least one or more of the following B1)-B6):
[0024] B1) The 17th amino acid residue is mutated from a threonine residue to a serine residue;
[0025] B2) The 56th amino acid residue is mutated from a leucine residue to a valine residue;
[0026] B3) The 68th amino acid residue is mutated from an asparagine residue to an aspartic acid residue;
[0027] B4) The 77th amino acid residue is mutated from an isoleucine residue to a valine residue;
[0028] B5) The 135th amino acid residue is mutated from a serine residue to a glycine residue;
[0029] The 219th amino acid residue in B6 is mutated from a serine residue to a valine residue.
[0030] Preferably, the amino acid residue site mutations also include all mutations in B1-B6.
[0031] Preferably, the TEV protease mutant comprises the amino acid sequence shown in SEQ ID NO.3; preferably, the nucleic acid molecule encoding the protease mutant comprises the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.4.
[0032] It is understandable that those skilled in the art can select appropriate gene editing systems and methods to modify the mutants described above, based on the actual circumstances.
[0033] On the other hand, this application also provides biological materials, said biological materials comprising any one of the following C1)-C5):
[0034] C1) A nucleic acid molecule, wherein the nucleic acid molecule contains a nucleic acid molecule encoding the TEV protease mutant described herein;
[0035] C2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in C1);
[0036] C3) A recombinant vector containing the nucleic acid molecule described in C1) and / or the expression cassette described in C2);
[0037] C4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3);
[0038] C5) Recombinant cells, wherein the recombinant cells contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3).
[0039] The expression cassette described herein may also include functional elements such as promoters, terminators, and marker genes. Those skilled in the art can make conventional selections according to the actual situation, as long as the expression of C1 nucleic acid molecules can be completed. No further restrictions are placed on the structure and composition of the expression cassette here.
[0040] The vectors described herein refer to those capable of delivering exogenous DNA or target genes into host cells for amplification and expression. These vectors can be any vector (e.g., plasmids or viruses) that facilitates recombinant DNA manipulation and the expression of nucleic acid sequences. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed-circular plasmids. Vectors can be self-replicating vectors (i.e., complete structures existing outside the chromosome that can replicate independently of the chromosome), such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes. Vectors can contain any mechanism that ensures self-replication. Alternatively, a vector is one that, upon introduction into a host cell, integrates into the genome and replicates along with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, may be used, as those skilled in the art can choose according to the specific circumstances; no excessive limitations are imposed here.
[0041] In a preferred embodiment, the recombinant vector may be a pRK plasmid. Preferably, the plasmid has an N-terminus linked to an MBP-TEV-HIS tag and a C-terminus linked to a 5R lysis-promoting tag.
[0042] Furthermore, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO.18 or a nucleotide sequence having at least 95% or more identity with SEQ ID NO.18.
[0043] More preferably, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO. 18 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 18.
[0044] Furthermore, the recombinant microorganism is one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and Saccharomyces cerevisiae.
[0045] More preferably, the recombinant microorganism is Escherichia coli.
[0046] In one specific embodiment, the recombinant microorganism is Escherichia coli BL21(DE3).
[0047] Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can be used as the starting strain, as long as they can complete the expression of the TEV protease mutant described in this application. No specific strain is limited here.
[0048] In one optional embodiment, the expression cassette of the nucleic acid molecule is located on a recombinant vector or introduced into a recombinant microorganism using a recombinant vector. It should be noted that, as will be known to those skilled in the art, the nucleic acid molecule can be selectively inserted into the genome of the starting strain or can exist on a free plasmid, as long as the expression of the nucleic acid molecule or the synthesis of a TEV protease mutant can be achieved.
[0049] In one optional embodiment, the recombinant vector and recombinant microorganism contain an resistance selection marker gene. Those skilled in the art can choose according to the actual situation. This application does not impose mandatory limitations on the resistance selection marker gene.
[0050] A resistance selection marker gene is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or a protrophic auxotrophic phenotype. Examples of bacterial selection markers include the dal gene in Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline.
[0051] On the other hand, this application also provides the application of the aforementioned biomaterials in the preparation of TEV protease.
[0052] Preferably, the TEV protease is a TEV protease mutant.
[0053] More preferably, the amino acid sequence of the TEV protease mutant comprises a sequence obtained by mutating an amino acid residue site of the wild-type TEV protease, wherein the amino acid residue site is selected from one or more of positions 230, 40, 60, 176, and 217, and the wild-type TEV protease comprises the amino acid sequence shown in SEQ ID NO.1.
[0054] More preferably, the amino acid residue site mutation includes at least one or more of the following A1)-A6):
[0055] A1) The 230th amino acid residue is mutated from a glutamic acid residue (E) to a leucine residue (L);
[0056] A2) The 40th amino acid residue is mutated from a phenylalanine residue (F) to a tyrosine residue (Y);
[0057] A3) The 60th amino acid residue is mutated from a leucine residue (L) to a histidine residue (H);
[0058] A4) The 60th amino acid residue is mutated from a leucine residue (L) to a tryptophan residue (W);
[0059] A5) The 138th amino acid residue is mutated from isoleucine residue (I) to threonine residue (T);
[0060] A6) The 176th amino acid residue is mutated from an asparagine residue (N) to a valine residue (V);
[0061] The 217th amino acid residue in A7 is mutated from a phenylalanine residue (F) to a glutamine residue (Q).
[0062] On the other hand, this application also provides a method for preparing TEV protease, the method comprising: producing TEV protease by fermentation using the recombinant microorganism.
[0063] Preferably, the TEV protease is a TEV protease mutant.
[0064] More preferably, the amino acid sequence of the TEV protease mutant comprises a sequence obtained by mutating an amino acid residue site of the wild-type TEV protease, wherein the amino acid residue site is selected from one or more of positions 230, 40, 60, 176, and 217, and the wild-type TEV protease comprises the amino acid sequence shown in SEQ ID NO.1.
[0065] More preferably, the amino acid residue site mutation includes at least one or more of the following A1)-A6):
[0066] A1) The 230th amino acid residue is mutated from a glutamic acid residue (E) to a leucine residue (L);
[0067] A2) The 40th amino acid residue is mutated from a phenylalanine residue (F) to a tyrosine residue (Y);
[0068] A3) The 60th amino acid residue is mutated from a leucine residue (L) to a histidine residue (H);
[0069] A4) The 60th amino acid residue is mutated from a leucine residue (L) to a tryptophan residue (W);
[0070] A5) The 138th amino acid residue is mutated from isoleucine residue (I) to threonine residue (T);
[0071] A6) The 176th amino acid residue is mutated from an asparagine residue (N) to a valine residue (V);
[0072] The 217th amino acid residue in A7 is mutated from a phenylalanine residue (F) to a glutamine residue (Q).
[0073] Those skilled in the art will recognize that the recombinant microorganisms can be cultured and / or fermented using common methods.
[0074] In a preferred embodiment, the method for preparing the TEV protease includes the following steps:
[0075] Step 1: Construct the recombinant microorganism;
[0076] Step 2: Inoculate the recombinant microorganisms into the culture medium at an inoculum rate of 1%-10% and incubate at 25℃-40℃ and 100-300rpm for 1-5 hours until OD reaches 100%. 600 =0.6-0.8, add IPTG inducer to a final concentration of 0.1mM, and induce for 12-24h in a shaker at 25℃-40℃ and 100-300rpm.
[0077] On the other hand, this application also provides the use of the mutant or the biological material in increasing TEV protease yield and / or increasing TEV protease activity.
[0078] Preferably, the increase in TEV protease yield is achieved by increasing the expression level of soluble TEV protease, effectively avoiding intramolecular cleavage, and maintaining the activity and stability of the protease.
[0079] This application is the first to discover that mutations at sites F40Y, L60H, L60W, I138T, N176V, F217Q, and E230L of the TEV protease can effectively improve its expression level, especially the expression level of soluble protein, providing new functional sites for the study of TEV protease.
[0080] The present invention has the following beneficial effects:
[0081] This invention constructs different TEV protease mutants by introducing mutation sites into the TEV protease and discovers beneficial mutation sites that can directly increase expression levels. These include mutations at amino acid residues 230 (from glutamic acid to leucine), 40 (from phenylalanine to tyrosine), 60 (from leucine to histidine), 60 (from leucine to tryptophan), 138 (from isoleucine to threonine), 176 (from asparagine to valine), and 217 (from phenylalanine to glutamine). These mutations provide new genetic engineering sites and a research foundation for the study of TEV protease expression systems and the improvement of yield.
[0082] This invention utilizes a highly efficient *E. coli* protein expression system to express a TEV protease mutant. This TEV protease mutant not only possesses the functional activity and specificity of the natural enzyme but also avoids intramolecular cleavage, maintaining the protease's activity and stability. Furthermore, it exhibits a high soluble expression level of TEV protein, effectively reducing downstream purification costs. This provides a theoretical basis for the large-scale production of TEV protease. Attached Figure Description
[0083] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:
[0084] Figure 1 This is a schematic diagram of the structure of a protein expression plasmid. Detailed Implementation
[0085] Technical terms:
[0086] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.
[0087] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.
[0088] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.
[0089] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.
[0090] Recombinant microorganisms: bacterial cell lines in which foreign genes are expressed efficiently using genetic engineering methods.
[0091] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.
[0092] To more clearly illustrate the overall concept of this application, a detailed description is provided below by way of embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.
[0093] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. For embodiments where specific conditions are not specified, follow standard conditions or conditions recommended by the manufacturer. The plasmids, restriction enzymes, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercially available products, and specific operations were performed according to the kit instructions.
[0094] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).
[0095] In this specification, the amino acids at the corresponding sites are represented by the recognized IUPAC single-letter abbreviations, where each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).
[0096] In this specification, mutations in amino acids are indicated by "original amino acid, site, substituted amino acid". For example, the mutation of glutamic acid E at site 230, which is sequentially counted from the N (nitrogen) end to the C (carbon) end of the sequence, to leucine L is represented as E230L.
[0097] All plasmids used in this invention were designed using conventional molecular biology techniques, and constructed through PCR and vector fragment recombination. All recombinant plasmids were verified by sequencing to be completely consistent with the target sequence.
[0098] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.
[0099] In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage.
[0100] Example 1: Construction and Screening of TEV Protease Mutants
[0101] This embodiment provides a process for constructing and screening TEV protease mutants.
[0102] The gene sequence of the original TEV protease is derived from Tobacco etch virus, and its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.
[0103] Based on existing mutants with high enzyme activity, the original TEV protease was modified by mutating the following amino acid residues: threonine at position 17 to serine, leucine at position 56 to valine, asparagine at position 68 to aspartic acid, isoleucine at position 77 to valine, serine at position 135 to glycine, and serine at position 219 to valine, resulting in the TEV-01 protein. After codon optimization of the target gene, the TEV-01 strain was obtained through whole-genome synthesis. An MBP-TEV-HIS tag was attached to the N-terminus, and a 5R tag was attached to the C-terminus to improve the soluble expression of the target protein. Its amino acid sequence is shown in SEQ ID NO. 3, and its nucleotide sequence is shown in SEQ ID NO. 4.
[0104] For mutation methods, please refer to the literature: (1) Cesaratto F, Burrone RO, Petris G. Tobacco EtchVirus protease: A shortcut across biotechnologies[J]. Journal of Biotechnology, 2016, 231239-249. (2) Sergio E, Ignacio JMLMLD, Antonio LF, et al. Improvedyield, stability, and cleavage reaction of a novel tobacco etch virus proteasemutant.[J].Applied microbiology and biotechnology,2022,106(4):1-18.(3)HeejinN,JBH,Deog-Young C,et al.Tobacco etch virus(TEV)protease with multiplemutations to improve solubility and reduce self-cleavage exhibits enhancedenzymatic activity.[J].FEBS open bio,2020,10(4):619-626.
[0105] Based on this, the three-dimensional structure of the TEV-01 protein was obtained using an online server, and the surface amino acids of the TEV-01 protein were viewed using PyMOL visualization editing software. Single-point mutation experiments were performed on the following 11 amino acid sites. A pRK plasmid containing the TEV-01 gene (e.g., ...) was used. Figure 1 Using 11 amino acid sites as templates, the mutation primers shown in Table 1 were designed to perform single-point mutations at 11 sites in the TEV-01 amino acid sequence, and PrimeSTAR GXL was used. Full plasmid amplification was performed using Premix (purchased from Takara). After amplification, the PCR products were detected by agarose gel electrophoresis. Once the amplified band sizes were confirmed to be correct, the PCR products were detemplated using DpnI enzyme. The detemplated PCR products were then recovered. 20 μL of the recovered PCR product was transferred into thawed *E. coli* competent cells that had been placed on ice. After gentle mixing, the cells were incubated on ice for 30 min. A heat shock was then performed at 42°C for 60 seconds. After heat shock, the competent cells were immediately placed on ice and incubated for 2 min. 500 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C with shaking for 1 h. After recovery, 100 μL of the bacterial culture was evenly spread onto LB agar plates containing 100 μg / mL ampicillin. The plates were inverted and incubated overnight at 37°C for 12-16 h. Single colonies were picked from the plates after incubation and sent for sequencing. Plasmids were extracted from the correctly sequenced bacterial cultures to obtain the corresponding TEV mutant plasmids.
[0106] Table 1 Primers used for PCR amplification
[0107]
[0108]
[0109] Example 2: Expression and Detection of Mutant Protein
[0110] The TEV mutant plasmid obtained in Example 1 was transformed into Escherichia coli BL21(DE3) competent cells as follows: Escherichia coli BL21(DE3) competent cells were thawed on ice in advance, 5 μL of mutant plasmid was added, and the cells were incubated on ice for 30 min. Heat shock was performed accurately in a water bath at 42℃ for 60 s. After heat shock, the competent cells were quickly placed on ice and allowed to stand for 2 min. 500 μL of antibiotic-free LB medium was added, and the cells were thawed and cultured in a shaker at 37℃ for 1 h. After thaw, 100 μL of bacterial culture was evenly spread with glass beads on LB solid agar plates containing 100 μg / mL ampicillin. The plates were inverted and incubated overnight at 37℃ for 12 h to obtain engineered bacteria capable of expressing mutant proteins.
[0111] The method for detecting the expression level of TEV mutant protein is as follows: A single colony of the engineered bacteria expressing the mutant protein in good growth condition was picked and placed in 3 mL of LB liquid medium. The culture was incubated at 37℃ and 200 rpm for 4-5 h. Then, 200 μL of the bacterial culture was added to a shake flask containing 20 mL of LB liquid medium containing 100 μg / mL ampicillin. The culture was incubated at 37℃ and 200 rpm for 2-3 h until OD (Organic Degradation) was reached.600 =0.6-0.8, add IPTG inducer to a final concentration of 0.1 mM, and induce for 16 h in a shaker at 30℃ and 150 rpm. After induction, measure the OD of the bacterial culture using a UV spectrophotometer. 600 Collect 15 OD 600 Centrifuge the bacterial cells to remove the supernatant, add 2 mL of lysis buffer to resuspend the bacterial cells, sonicate at 25% power on ice for 5 min, and centrifuge the total enzyme solution after cell disruption to collect the supernatant, thus obtaining the supernatant of the corresponding mutant protein. The expression of mutant proteins was determined by SDS-PAGE gel electrophoresis. A 12% pre-prepared gel was loaded, and an appropriate amount of electrophoresis buffer was added above the minimum water level line of the electrophoresis tank. 20 μL of supernatant was added to 20 μL of 2×SDS loading buffer, mixed well, and heated in a 99℃ metal bath for 10-12 min to completely denature the proteins. The heated samples were then placed at room temperature for 10 min. 10 μL of Rainbow Maker and 10 μL of each sample were then loaded for SDS-PAGE gel electrophoresis at a constant voltage of 120V for 1 h 20 min. After electrophoresis, the gel was removed, and the PAGE gel was placed in a culture dish with an appropriate amount of Coomassie Brilliant Blue staining solution. The mixture was stained at room temperature on a shaker at 75 rpm for 2 h. The staining solution was recovered, and an appropriate amount of destaining solution was added for overnight destaining. After destaining, the gel was photographed and analyzed using a gel imaging system.
[0112] The expression levels of the different TEV mutant proteases constructed above are compared in Table 2. The TEV mutant strains with increased expression levels that were successfully screened include F40Y (amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.6), L60H (amino acid sequence as shown in SEQ ID NO.7, nucleotide sequence as shown in SEQ ID NO.8), L60W (amino acid sequence as shown in SEQ ID NO.9, nucleotide sequence as shown in SEQ ID NO.10), I138T (amino acid sequence as shown in SEQ ID NO.11, nucleotide sequence as shown in SEQ ID NO.12), N176V (amino acid sequence as shown in SEQ ID NO.13, nucleotide sequence as shown in SEQ ID NO.14), F217Q (amino acid sequence as shown in SEQ ID NO.15, nucleotide sequence as shown in SEQ ID NO.16), and E230L (the 230th amino acid residue is mutated from glutamic acid to leucine, amino acid sequence as shown in SEQ ID NO.17, nucleotide sequence as shown in SEQ ID NO.18). The mutant M11 increased the soluble expression level of TEV protease by 39%.
[0113] Table 2 Comparison of mutant numbers, mutation sites, and relative expression levels of soluble proteins in Implementation Case 1
[0114] Mutant number mutation site Relative expression level (%) TEV-01 \ 100 M1 S17C 88 M2 F40Y 116 M3 L60H 119 M4 L60W 114 M5 I138T 106 M6 N176V 112 M7 V182I 98 M8 Q197E 87 M9 H214L 47 M10 F217Q 110 M11 E230L 139
[0115] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.
Claims
1. A mutant of TEV protease characterized in that, The amino acid sequence of the TEV protease mutant comprises a sequence obtained by mutating amino acid residue sites of the wild-type TEV protease, wherein the amino acid residue sites are selected from one or more of positions 230, 40, 60, 138, 176, and 217, and the wild-type TEV protease comprises the amino acid sequence shown in SEQ ID NO.
1.
2. The mutant according to claim 1, characterized in that, The amino acid residue site mutation includes at least one or more of the following A1)-A6): A1) The 230th amino acid residue is mutated from a glutamic acid residue to a leucine residue; A2) The 40th amino acid residue is mutated from a phenylalanine residue to a tyrosine residue; A3) The 60th amino acid residue is mutated from a leucine residue to a histidine residue; A4) The 60th amino acid residue is mutated from a leucine residue to a tryptophan residue; A5) The 138th amino acid residue is mutated from an isoleucine residue to a threonine residue; A6) The amino acid residue at position 176 is mutated from an asparagine residue to a valine residue; The 217th amino acid residue in A7 is mutated from a phenylalanine residue to a glutamine residue.
3. The mutant according to any one of claims 1-3, characterized in that, The TEV protease mutant is obtained by mutating the glutamic acid residue at position 230 of the wild-type TEV protease to a leucine residue; preferably, the TEV protease mutant includes the amino acid sequence shown in SEQ ID NO.
17.
4. A biomaterial, characterized in that, The biomaterial includes any one of the following C1)-C5): C1) A nucleic acid molecule, wherein the nucleic acid molecule contains a nucleic acid molecule encoding a TEV protease mutant according to any one of claims 1-3; C2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) and / or the expression cassette described in C2); C4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3); C5) Recombinant cells, wherein the recombinant cells contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3).
5. The biomaterial according to claim 4, characterized in that, The nucleic acid molecule includes a nucleotide sequence as shown in SEQ ID NO.18 or a nucleotide sequence having at least 95% or more identity with SEQ ID NO.
18.
6. The biomaterial according to claim 5, characterized in that, The recombinant microorganism is one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and Saccharomyces cerevisiae.
7. The use of the biomaterials as described in any one of claims 5-6 in the preparation of TEV protease.
8. A method for preparing TEV protease, characterized in that, The method includes: producing TEV protease by fermentation using the recombinant microorganisms described in claim 5.
9. The use of the mutants as described in any one of claims 1-3 or the biological materials as described in any one of claims 4-6 in increasing TEV protease yield and / or increasing TEV protease activity.