Tev protease mutants, methods of making and using same

Abstract

This invention relates to the field of genetic engineering technology, and particularly to TEV protease mutants, their preparation methods, and applications. This invention modifies the TEV enzyme molecule, with mutation points differing from those reported in previous studies. This enhances the activity of the TEV protease at the molecular level. Furthermore, this invention utilizes CRISPR / Cas9 gene editing techniques to modify the host bacterial chassis, obtaining engineered strains that express TEV at high yields. Further, the provided high-density fermentation control method can rapidly promote TEV enzyme expression, resulting in high E. coli cell yield, with an OD600 value reaching approximately 150, a cell wet weight of 80–130 g / L, and high expression levels of the target protein, accounting for 40%–64% of the total bacterial protein. Simultaneously, the method provided by this invention is simple and easy to implement, using readily available raw materials, achieving good application results while reducing production costs, resulting in significant economic benefits.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to TEV protease mutants, their preparation methods, and applications. Background Technology

[0002] TEV enzyme (Tobacco Spot Virus Protease) is an Nla protease derived from tobacco spot virus. It exhibits strong site specificity, specifically recognizing the sequence E-Xaa-Xaa-Y-Xaa-QG / S, with the most commonly used sequence being Glu-Asn-Leu-Tyr-Phe-Gln-Gly (or ENLYFQG). Its cleavage site is between the last two amino acids, leaving only one amino acid residue at the N-terminus of the target protein. TEV enzyme's sequence specificity is far higher than that of proteases such as thrombin, factor Xa, and enterokinase. Furthermore, this enzyme has a wide range of reaction conditions, exhibiting catalytic activity within a pH range of 5.5-8.5 and a temperature range of 4-30℃.

[0003] It is precisely because of these excellent properties of TEV enzymes that they have been widely used in the field of protein research. Whether it is the in-depth analysis of protein structure or the exploration and discovery of protein function, TEV enzymes play an indispensable role and have become a powerful tool in the hands of many researchers, continuously contributing to the advancement of protein research.

[0004] However, the expression and purification of wild-type TEV enzymes in *E. coli* inevitably suffer from several drawbacks. First, severe intramolecular self-cleavage leads to instability in the enzyme's molecular structure and abnormal functional changes, making it difficult to accurately function in subsequent applications. Second, a large amount of wild-type TEV enzyme exists as inclusion bodies, which significantly reduces its effective content in the active state. Furthermore, the processing and conversion of inclusion bodies into active enzyme molecules requires complex and cumbersome steps, increasing production and time costs. Third, its yield is quite limited, failing to meet the needs of large-scale scientific research and industrial production, often resulting in supply shortages in practical applications. In addition, the low water solubility of TEV enzymes greatly limits their application in various reaction systems, as many reactions require aqueous solutions, and low water solubility affects the sufficient contact and effective reaction between the enzyme and substrate.

[0005] These combined shortcomings severely limit the application scope of wild-type TEV enzymes from multiple dimensions, making them difficult to utilize effectively in a wider range of fields and more diverse experimental and production scenarios. To address these issues, some existing technologies select suitable leader peptides to help TEV enzymes fold correctly and achieve soluble expression, while others improve their preparation methods. However, although these methods have solved the problems of low water solubility, poor activity, and stability of TEV enzyme expression to some extent, they have not solved the problem of large-scale TEV production. Specifically, insufficient research has been conducted on the expression host strain and how to achieve high-density fermentation for TEV enzyme production. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a TEV protease mutant, its preparation method and application.

[0007] The present invention provides a mutant of tobacco etch virus cysteine ​​protease, wherein the amino acid sequence of the tobacco etch virus cysteine ​​protease is shown in SEQ ID NO:1, and the mutant includes at least one of the following mutation sites:

[0008] T17S, L56V, N68D, I77V, S135G, W143N, M121P, A195K, L210N, M218A.

[0009] In some embodiments, the mutation sites of the mutant include T17S, L56V, N68D, I77V, S135G and at least one of the following: W143N, M121P, A195K, L210N, M218A.

[0010] In this invention, the cysteine ​​protease shown in SEQ ID NO:1 is derived from tobacco etch virus. This invention utilizes a previously disclosed TEV mutant of the tobacco etch virus cysteine ​​protease (denoted as M0, containing mutation sites T17S, L56V, N68D, I77V, and S135G) and further modifies it molecularly.

[0011] In a specific embodiment, the mutation sites of the mutant include:

[0012] T17S, L56V, N68D, I77V, S135G and W143N;

[0013] Or including T17S, L56V, N68D, I77V, S135G and M121P;

[0014] This may include T17S, L56V, N68D, I77V, S135G, and A195K;

[0015] Or including T17S, L56V, N68D, I77V, S135G and L210N;

[0016] Or include T17S, L56V, N68D, I77V, S135G and M218A;

[0017] Or including T17S, L56V, N68D, I77V, S135G, W143N and M121P;

[0018] Or including T17S, L56V, N68D, I77V, S135G, W143N and A195K;

[0019] Or including T17S, L56V, N68D, I77V, S135G, W143N and L210N;

[0020] Or including T17S, L56V, N68D, I77V, S135G, W143N and M218A;

[0021] Or including T17S, L56V, N68D, I77V, S135G, M121P and A195K;

[0022] Or including T17S, L56V, N68D, I77V, S135G, M121P and L210N;

[0023] Or including T17S, L56V, N68D, I77V, S135G, M121P and M218A;

[0024] Or including T17S, L56V, N68D, I77V, S135G, A195K and L210N;

[0025] Or including T17S, L56V, N68D, I77V, S135G, A195K and M218A;

[0026] Or including T17S, L56V, N68D, I77V, S135G, L210N and M218A;

[0027] Or including T17S, L56V, N68D, I77V, S135G, W143N, M121P and A195K;

[0028] Or including T17S, L56V, N68D, I77V, S135G, W143N, M121P and L210N;

[0029] Or including T17S, L56V, N68D, I77V, S135G, W143N, M121P and M218A;

[0030] Or including T17S, L56V, N68D, I77V, S135G, W143N, A195K and L210N;

[0031] Or including T17S, L56V, N68D, I77V, S135G, W143N, A195K and M218A;

[0032] Or including T17S, L56V, N68D, I77V, S135G, W143N, L210N and M218A;

[0033] Or including T17S, L56V, N68D, I77V, S135G, M121P, A195K and L210N;

[0034] Or including T17S, L56V, N68D, I77V, S135G, M121P, A195K and M218A;

[0035] Or including T17S, L56V, N68D, I77V, S135G, M121P, L210N and M218A;

[0036] Or including T17S, L56V, N68D, I77V, S135G, A195K, L210N and M218A;

[0037] Or including T17S, L56V, N68D, I77V, S135G, M121P, A195K, L210N and M218A;

[0038] Or including T17S, L56V, N68D, I77V, S135G, W143N, A195K, L210N and M218A;

[0039] Or including T17S, L56V, N68D, I77V, S135G, W143N, M121P, L210N and M218A;

[0040] Or including T17S, L56V, N68D, I77V, S135G, W143N, M121P, A195K and M218A;

[0041] Or including T17S, L56V, N68D, I77V, S135G, W143N, M121P, A195K and L210N;

[0042] Or include T17S, L56V, N68D, I77V, S135G, W143N, M121P, A195K, L210N and M218A.

[0043] More specifically, the mutation sites in the mutant include:

[0044] T17S, L56V, N68D, I77V, S135G, M121P, W143N, and A195K;

[0045] Or including T17S, L56V, N68D, I77V, S135G, M121P, W143N, A195K and L210N;

[0046] Or including T17S, L56V, N68D, I77V, S135G, W143N, A195K and M218A;

[0047] Or including T17S, L56V, N68D, I77V, S135G, M121P, W143N, A195K, L210N; and M218A;

[0048] This may include T17S, L56V, N68D, I77V, S135G, A195K, and L210N.

[0049] The TEV enzyme provided by this invention has good enzyme activity, and its cleavage activity can reach 100% in 4 hours.

[0050] The present invention also provides a nucleic acid encoding the mutant described herein.

[0051] This invention optimizes the TEV protease (M0) gene sequence according to E. coli codons, and further modifies it to obtain the nucleic acid sequence encoding the mutant described in this invention. Preferably, the codon encoding M121P is ccg, the codon encoding W143N is aac, the codon encoding A195K is aaa, the codon encoding L210N is aac, and the codon encoding M218A is gcg.

[0052] In some embodiments, the nucleic acid sequence in this invention is as shown in any one of SEQ ID NO: 2 to 6;

[0053] Or it has a sequence that is based on a nucleic acid sequence as shown in any one of SEQ ID NO:2 to 6, by substitution, deletion, addition and / or replacement of one or more nucleotides;

[0054] Or a sequence having more than 80% homology with the nucleic acid sequence shown in any one of SEQ ID NO:2 to 6.

[0055] The homology of 80% or more includes 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0056] Furthermore, the present invention also provides an expression unit comprising a promoter and nucleic acid as described above.

[0057] In this invention, the expression unit may also include a terminator and / or an enhancer. In some embodiments, the promoter is a eukaryotic promoter or a prokaryotic promoter, which is not limited in this invention. For example, the promoter is the PGK promoter, U6CAG promoter, EF1a promoter, H1 promoter, CMV promoter, EFS promoter, CBh promoter, SFFV promoter, MSCV promoter, SV40 promoter, UBC promoter, or TRE promoter. For example, the enhancer is selected from the SV40 enhancer, SV-1 enhancer, CAGG enhancer, COPIA enhancer, ROSA26 enhancer, EF1A enhancer, PGK enhancer, EF1α enhancer, CMV enhancer, HARE5 enhancer, UBC enhancer, or ACT5C enhancer. For example, the terminator is selected from λ phage terminator, SV40 terminator, rrnB terminator, T7 phage terminator, CMV terminator, T0 phage terminator, bGH terminator, hGH terminator, or rbGlob terminator.

[0058] Furthermore, the present invention also provides a plasmid vector comprising the nucleic acid or expression unit as described above.

[0059] As previously mentioned, the vector containing the nucleic acid or the expression unit is a cloning vector, an expression vector, or a viral vector. In this invention, the vector is used for the storage, amplification, or expression of the nucleic acid or the expression unit, and this invention does not limit its use. In some embodiments, the vector is a plasmid vector, including pET series plasmid vectors, pUC series plasmid vectors, pBR322 plasmid vectors, pGEM series plasmid vectors, Yeast series plasmid vectors, or Gateway plasmid vectors, etc. In some embodiments, pET series plasmid vectors are used to express the fusion protein. As feasible examples, the pET series expression vectors are selected from: pET-28a, pET-28a-EBFP, pET-5a(+), pET-5b, pET-5c, pET28a-ECFP, pET-9a, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-22b, pET-23a, pET-24a, or pET-25b. In some specific embodiments, the backbone vector of the plasmid vector is pET28a.

[0060] Furthermore, the present invention also provides a transformant that is transformed or transfected with the plasmid vector as described above, or whose genome integrates the nucleic acid as described above or the expression unit as described above.

[0061] In this invention, the host is a eukaryotic or prokaryotic host. The eukaryotic host includes, but is not limited to, yeast, insect cells, and renal epithelial cells, while the prokaryotic host includes, but is not limited to, *Escherichia coli*. In some embodiments, the host of the transformant described in this invention is *Escherichia coli*.

[0062] In this embodiment of the invention, the host is Escherichia coli DH5α strain, BL21(DE3) strain, BL21(DE3)pLysS strain, Rosetta strain, JM109 strain, JM110 strain, TOP10 strain, HB101 strain or Xl1-Blue strain.

[0063] In some embodiments, the aldA and / or cspD genes in the *E. coli* are knocked out.

[0064] In some specific embodiments, the *E. coli* strain is *E. coli* BL21(DE3) or *E. coli* Rosetta. More specifically, the *E. coli* strain is a BL21(DE3) strain with the aldA and / or cspD genes knocked out, or a Rosetta strain with the aldA and / or cspD genes knocked out.

[0065] Furthermore, the present invention also provides a method for preparing the mutant as described above, comprising: culturing the transformant as described above, and inducing the expression of the mutant.

[0066] In some embodiments, the culture medium comprises water and magnesium sulfate heptahydrate 1-5 g / L, dipotassium hydrogen phosphate 5-15 g / L, potassium dihydrogen phosphate 1-10 g / L, citric acid monohydrate 1-5 g / L, ammonium sulfate 1-5 g / L, yeast extract 1-10 g / L, and trace elements 0.1-1 mL / L.

[0067] Specifically, the culture medium includes water and magnesium sulfate heptahydrate 2 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 5 g / L, citric acid monohydrate 3 g / L, ammonium sulfate 3 g / L, yeast extract 5 g / L, and trace elements 1 mL / L.

[0068] The trace elements are a mother liquor containing trace elements, and each liter of the trace element mother liquor includes water and EDTA sodium salt, 15.0 mg; ZnSO4·7H2O, 0.45 mg; CoCl2·6H2O, 0.3 mg; MnCl2·2H2O, 1 mg; H3BO3, 0.1 mg; CuSO4·5H2O, 0.3 mg; FeSO4·7H2O, 0.3 mg; Na2MoO4·2H2O, 0.4 mg; and KI, 0.1 mg (per liter).

[0069] In some embodiments, the culture conditions include:

[0070] The pH is 6.8–7.0, the temperature is 37℃, the tank pressure is 0.04–0.06 MPa, the rotation speed is 200 rpm, the air volume is 2–3 L / min, and after culturing for 4–5 h, the dissolved oxygen is controlled by fed-batch feeding at 30%–55%. After culturing until the OD600 is 18–52, IPTG is added to a final concentration of 0.1–0.5 mM, and the temperature is lowered to 25–30℃ for induction for 12 h.

[0071] More specifically,

[0072] The pH value of the culture is 6.8, 6.9, or 7.0. The initial culture temperature is 35–40°C, for example, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0073] The culture tank pressure is 0.04 MPa, 0.05 MPa, or 0.06 MPa.

[0074] The rotation speed of the culture is 100 to 300 rpm, for example, 100 rpm, 110 rpm, 120 rpm, 150 rpm, 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 280 rpm or 300 rpm.

[0075] The wind speeds used for cultivation were 2 L / min, 2.2 L / min, 2.5 L / min, 2.8 L / min, 2.9 L / min, and 3.0 L / min.

[0076] The dissolved oxygen level is controlled to be 30% to 55%, for example, 30%, 35%, 40%, 45%, 50%, or 55%.

[0077] The OD600 values ​​are 18, 19, 20, 21, 22, 23, 24, 25, 29, 30, 32, 35, 38, 40, 41, 45, 49, 50, 51, or 52.

[0078] The final concentration of IPTG is 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM or 0.5 mM.

[0079] The temperature is lowered to 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C for induction.

[0080] The induction duration is 8 to 16 hours, for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or 16 hours.

[0081] In some specific embodiments, the culture is carried out until the OD600 is 28-32, IPTG is added to a final concentration of 0.3mM, and the temperature is lowered to 28°C for induction for 12 hours.

[0082] Furthermore, the present invention also provides the application of the aforementioned mutants, nucleic acids as described above, expression units as described above, plasmid vectors as described above, transformants as described above, or products prepared by the aforementioned methods in the preparation of protease formulations.

[0083] In this invention, the recognition sequence of the protease is ENLYFQG, and the restriction site is between Q and G.

[0084] Furthermore, the present invention also provides a protease preparation comprising the mutant described above, the nucleic acid as described above, the expression unit as described above, the plasmid vector as described above, the transformant as described above, or the product obtained by the preparation method as described above.

[0085] The protease preparation described in this invention also includes a buffer solution required for enzymatic hydrolysis. This invention does not limit the composition of the buffer solution. For example, the buffer solution may be Tris-HCl buffer, HEPES buffer, or phosphate-buffered saline (PBS).

[0086] Furthermore, the present invention also provides a method for protease digestion, comprising treating a protein with a protease preparation as described above, wherein the protein is a protein containing the ENLYFQG fragment.

[0087] In this invention, the enzymatic digestion reaction system includes the protein to be digested and 50mM Tris-HCl (pH 8.0), 50mM NaCl, 2mM MTT, and 1mM EDTA.

[0088] This invention modifies the TEV enzyme molecule, with mutations differing from previous reports. This enhances the activity of the TEV protease at the molecular level. Furthermore, this invention utilizes CRISPR / Cas9 gene editing to modify the host bacterial chassis, obtaining an engineered strain that highly expresses TEV. Moreover, the provided high-density fermentation control method rapidly promotes TEV enzyme expression, resulting in high E. coli cell yield, an OD600 value of approximately 150, a cell wet weight of 80–130 g / L, and high expression levels of the target protein, which accounts for 40%–64% of the total bacterial protein. Simultaneously, the method provided by this invention is simple and easy to implement, using readily available raw materials, achieving good application results while reducing production costs, resulting in significant economic benefits. Attached Figure Description

[0089] Figure 1 Enzyme digestion activity results: Substrate molecular weight: 65.9kDa (top band), digested product: 52.3kDa (bottom band), activity was calculated based on the proportion of the small molecular weight product generated by digestion, of which: (1): TEV-M0, (2): TEV-M1, (3): TEV-M2, (4): TEV-M3, (5): TEV-M4, (6): TEV-M5;

[0090] Figure 2 .aldA gene knockout verification;

[0091] Figure 3 .cspD gene knockout verification;

[0092] Figure 4 Comparison of TEV-M3 expression in different engineered strains, where: A: host strain is BL21(DE3), B: Rosetta, C: BL21(ΔaldA), D: BL21(ΔcspD);

[0093] Figure 5.TEV-M3 was screened by fermentation in a 15L fermenter with different engineered strains;

[0094] Figure 6 The results of the enzymatic digestion reaction of TEV-BL21(ΔaldA) enzyme obtained by fermentation 2. Detailed Implementation

[0095] This invention provides TEV protease mutants, their preparation methods, and applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0096] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0097] Furthermore, unless otherwise stated herein, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, unless otherwise expressly indicated, the singular forms “a” and “this” include plural indicators.

[0098] In this application, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions "consisting of...".

[0099] In this application, “and / or” as used herein includes the meaning of “and”, “or”, and “all or any other combination of elements linked by the term”.

[0100] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0101] The TEV protease described in this invention is a cysteine ​​protease derived from Tobacco Etch Virus (TEV). Based on a publicly available mutant containing five mutation points, designated TEV-M0 in this paper, it was modified using this mutant as a template. The following is the specific mutation site information (TEV-M1; TEV-M2; M3, M4, M5). The mutated sites are marked with underscores.

[0102] Table 1. Amino acid sequences of enzymes

[0103]

[0104]

[0105] Table 2. Encoding nucleic acid sequences of the enzymes

[0106]

[0107]

[0108]

[0109]

[0110] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0111] The present invention will be further illustrated below with reference to the embodiments:

[0112] Example 1. Construction of expression vector 28a-TEV

[0113] The TEV protease gene sequence was optimized based on the E. coli codons and directly synthesized by Genscript Biotech Co., Ltd. The plasmid PET-28a was obtained by inserting NdeⅠ and HindⅢ into the E. coli expression vector PET-28a. 5 μL of the expression plasmid was transformed into BL21(DE3) competent cells, incubated on ice for 30 min, then heat-shocked at 42℃ for 1 min, and incubated on ice for another 3 min. 500 μL of antibiotic-free LB liquid medium was added, and the cells were cultured on a shaker at 37℃ and 200 rpm for 60 min. The culture was then spread on LB plates containing 50 μg / mL kanamycin and incubated overnight at 37℃ to obtain the expression strain.

[0114] Example 2. Mutation Construction and Screening

[0115] Using the TEV mutant already published in the literature as a comparative example (TEV = -M0, containing five mutation sites: T17S, L56V, N68D, I77V, S135G), further molecular modifications were performed on this parent strain to obtain proteases with higher properties. Using TEV-M0 enzyme as the parent, an AI-assisted rational design was conducted. The Evcouplings algorithm (protein mutation fitness prediction) was used to calculate the effect of single-point mutations at the evolutionary level, while the ThermoMPNN algorithm (protein thermostability mutation prediction) was used to calculate the effect of mutations on the structural thermostability of the TEV enzyme (PDB 1LVM). After normalization, the prediction results of the two algorithms were weighted and ranked, and the top 20 mutants with the highest scores were selected. Based on experience, obviously unreasonable predictions, such as those that disrupt the original hydrogen bond network or change active residues, were removed. Mutations that could fill structural cavities and replace hydrophobic residues with hydrophilic residues were preferred. Five rationally designed mutants were selected: W143N, M121P, A195K, L210N, and M218A. Then, these five mutations were stacked, and the complex structure model of the stacked mutant and the substrate ENLYFQG was generated using the AlphaFold2 structure prediction algorithm. The structures with the highest scores in pLDDT and ipTM were selected, and Gromacs was used for energy optimization and MM / GBSA affinity prediction. The results showed that the affinity of five stacked mutants was superior to that of the TEV-M0 enzyme in this patent. These five combinations with the highest scores were selected for molecular construction and activity screening experiments. 2.1 Mutant Construction:

[0116] Table 3. Primer List

[0117]

[0118]

[0119] Table 4. Constructing the Fragment List

[0120]

[0121] 2.1.1 PCR Cloning to Construct Mutants

[0122] PCR reaction system:

[0123] Prepare the reaction system according to the following formula:

[0124] Table 5

[0125] primerPF (10µM) 1ul primerPR (10µM) 1ul Template(A0A6P1) 0.5ul (20ng / ul) 5XQ5reactionBuffer 5ul 10mMdNTP 0.5ul Q5 polymerase 0.3ul <![CDATA[ddH2O]]> upto25ul

[0126] PCR loading system:

[0127] Perform amplification in a PCR instrument according to the following procedure:

[0128] Table 6

[0129]

[0130] After PCR is completed, the PCR products are run on gel electrophoresis, and the PCR-positive products are purified by gel extraction.

[0131] Homologous recombination:

[0132] Recombination and fusion of gene fragments from each mutant:

[0133] Table 7

[0134] PCR fragment F1 (100 ng / ul) 1μl 1μl PCR fragment F2 (100 ng / ul) 1μl 1μl PCR fragment F3 (100 ng / ul) / 1μl PCR fragment F4 (100 ng / ul) / 1μl 2xseamlesscloningMix 2μl 3μl

[0135] Reaction conditions: 50℃ for 30 min

[0136] 2.1.2 Transformation: BL21(DE3) was transgenic for expression to preserve the strain.

[0137] Take the homologous recombination product and transform it into 40 μL of BL21(DE3) competent cells. After incubating on ice for 30 min, heat shock at 42°C for 45 s, incubate on ice for 2-3 min, add 500 μL of SOC, and culture on a shaker for 1.5 h. Centrifuge, spread on plates, and incubate overnight at 37°C for 12-16 h. Select clones with high expression levels and preserve them at -80°C.

[0138] 2.2 Screening for mutant activity:

[0139] 2.2.1 Shake Flask Culture: Take the seed culture from a -80°C freezer and streak it onto a plate. The next day, pick colonies from the streaked plate and incubate them in 15 mL LB medium (50 mg / mL Kan antibiotic) at 37°C and 220 rpm. After 5 hours, transfer the culture to a shake flask containing 600 mL LB medium and continue incubation. When the OD600 reaches 0.8, cool to 25°C, add 0.3 mM IPTG for induction, and incubate overnight for 15 hours. Centrifuge to collect the bacterial cells.

[0140] 2.2.2 Purification: Seven protein samples, SUMO-ENLYFQG-A007, TEV-M0, TEV-M1, TEV-M2, M3, M4, and M5.

[0141] Take 2g of bacterial cells, homogenize by high pressure at 800 bar for 5 min. Purify by Ni column using the following steps: Prepare buffer A: 50mM Tris-HCl (pH 8.0), 300mM NaCl, 1mM DTT, 10mM imidazole. Prepare buffer B: 50mM Tris-HCl (pH 8.0), 300mM NaCl, 1mM DTT, 500mM imidazole. Use a protein purification instrument for sample loading, equilibration, washing (50mM imidazole), and elution (500mM imidazole). Confirm the collection of the correct target protein by gel electrophoresis, dialyze, and store at 4℃ for later use.

[0142] Test protein concentration:

[0143] 4.05 mg / ml, 2.71 mg / ml, 2.40 mg / ml, 3.15 mg / ml, 2.59 mg / ml, 2.12 mg / ml, 3.56 mg / ml. Corresponding molar concentrations: 61 μM, 90 μM, 80 μM, 105 μM, 86 μM, 71 μM, 119 μM.

[0144] 2.2.3 Testing protein activity:

[0145] To a 750 μL reaction system consisting of 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 2 mM MTT, and 1 mM EDTA, add 250 μL of the experimental substrate SUMO-ENLYFQG-A007, resulting in a final concentration of 15 μM. Before the reaction begins, add different volumes of the mutant TEV, maintaining the same final concentration of 0.5 μM. The prepared 1 mL reaction system was incubated at 30 °C and 200 rpm, with samples taken at 0 h, 1 h, 2 h, 4 h, and 6 h. (Gel coating: 20 μL sample + 5 μL of 5x Loading buffer.)

[0146] The SUMO-TEV recognition sequence-protein A (A007) structure: SUMO-ENLYFQG-A007 was used as a substrate to test the cleavage activity of different TEV mutants. A mutant already published in the literature was used as a comparative example, labeled TEV-M0. Substrate molecular weight: 65.9 kDa (top band), cleavage product (A007): 52.3 kDa (bottom band). Results are as follows... Figure 1 The statistical results are shown in Table 8:

[0147] Table 8. Statistics on cleavage activity of mutants (based on the percentage of products successfully cleaved by visual inspection)

[0148]

[0149] Example 3: Construction of knockout strains

[0150] To increase the expression of foreign proteins, the aldehyde dehydrogenase A gene (aldA) and the cold shock protein-like protein (DNA replication inhibitor) gene (cspD) of *E. coli* BL21(DE3) were knocked out. By knocking out some unnecessary metabolic pathways in the chassis cells, unnecessary metabolic consumption was reduced and DNA replication was increased, thereby increasing the expression of the exogenous target protein.

[0151] Build process:

[0152] First, sgRNA and donor plasmids were constructed: sgRNA was designed using the website: http: / / www.rgenome.net / cas-designer / . Then, the backbone was amplified, the plasmid was eliminated with DpnI, treated with SEAMLESS ligase, and transformed into DH5α. Single clones were selected for verification, and correct clones were preserved. Correct clones were expanded and cultured, and plasmids were extracted and stored for later use. Next, the aldA and cspD genes were located in the BL21(DE3) genome, and 500 bp upstream and downstream of each gene were selected as donors. Using BL21(DE3) cells as templates, the upstream and downstream 500 bp were amplified and purified by column chromatography. The upstream and downstream donors were fused by OEPCR, recovered by gel chromatography, and the plasmids were stored for later use.

[0153] Then, the pGRB-aldA,cspD plasmids were transformed into BL21(DE3). The specific steps were as follows: BL21(DE3) bacterial culture containing pECcas9 plasmid was streaked on an LB (kan) plate; then, a single colony was picked and cultured overnight in 5 ml of LB (kan) plate.

[0154] Transfer 500 μl of the bacterial culture to 50 ml of LB (kan) and incubate for 1-2 h until the OD reaches 0.1. Add arabinose to a final concentration of 10 mM (1 M arabinose, 500 μl in 50 ml); incubate for another 1-2 h until the OD is approximately 0.5-0.6, and begin preparing competent cells. Pre-chill the refrigerated centrifuge at 4°C, sterilize with 10% glycerol and sterile water, and then pre-chill at 4°C. Aliquot the bacterial culture into sterile centrifuge tubes and centrifuge at 6000 rpm for 3 min to collect the cells. Resuspend the cells in 40 ml of sterile water wash buffer in each tube and centrifuge at 6000 rpm for 3 min to collect the cells. Discard the supernatant, resuspend the cells in 40 ml of 10% glycerol wash buffer in each tube, and centrifuge at 6000 rpm for 3 min to collect the cells.

[0155] Resuspend each cell in 3 ml of 10% glycerol and aliquot 100 μl into 1.5 ml EP tubes. Add 100 ng of pGRB-gRNA plasmid and 200 ng of donor DNA to 100 μl of competent cells. Transform the cells by electroporation in a pre-chilled 1 mm electroporator (1.85 kV). Immediately after electroporation, resuspend the cells in 1 ml of fresh LB medium. Incubate the cells at 37°C for 2 hours, then spread all cells onto LB plates containing kanamycin (50 μg / ml) and ampicillin (50 μg / ml) and incubate overnight at 37°C. The next day, pick single clones for P verification (successful knockout is 1000 bp) (wild-type is the control). Cells with matching bands are sent for testing. Finally, the successfully knocked-out strain was transferred to 500 μl of LB agar (kan, final concentration 10 mM rhamnose (1 M rhamnose, 5 μl added to 500 μl of bacterial culture)) and cultured overnight. A loopful of bacterial culture was streaked onto an LB (kan) plate. Single colonies were picked and streaked onto LB (kan) and LB (amp) plates respectively. The growth on the plates was compared; the strain that grew on kan but not on amp was correct, indicating that the pGRB plasmid had been lost. The strain that lost the pGRB plasmid was inoculated into 2 ml of LB (antibiotic-free) and cultured overnight. A loopful of bacterial culture was streaked onto an LB (sucrose, 10 g / L) plate. Single colonies were picked and streaked onto LB (antibiotic-free) and LB (kan) plates respectively. The growth on the plates was observed; the strain that grew on the antibiotic-free plate but not on the kan plate was likely the strain that lost the pECcas9 plasmid. The strain that successfully lost the plasmid was expanded and cultured (LB, antibiotic-free) to preserve the strain. The validation results for the successful knockout of genes aldA and cspD can be found in [link to validation results]. Figure 2 , 3.

[0156] Example 4.

[0157] Transplantation to different host strains for expression and 15L fermentation screening: comparisons were made with conventional BL21(DE3), Rosetta, aldA knockout strains, and cspD knockout strains.

[0158] 4.1 Comparison of clonal expression levels among different strains (4 types):

[0159] To further screen for strains with good expression, the TEV-M3 plasmid was transfected into different host bacteria, and the expression was compared: TEV-M3 was expressed in both BL21(DE3) and Rosetta, and 10 clones were selected from each transfection experiment. TEV was transfected into BL21(ΔaldA) and BL21(ΔcspD). Clones transformed into BL21(ΔaldA) plates grew well, and 5 single clones were selected for protein expression, showing excellent expression levels. Clones transformed into BL21(ΔcspD) plates did not grow normally, and no large number of single clones appeared; 3 clones were selected for expression, but no expression was observed on the gel electrophoresis results.

[0160] 4.215L fermenter was used to ferment four types of bacterial strains, and a comparative experiment was conducted to show the expression of soluble strains.

[0161] Four types of engineered strains were screened by fermentation in a fermenter.

[0162] The fermentation medium consisted of 2 g / L magnesium sulfate heptahydrate, 10 g / L dipotassium hydrogen phosphate, 5 g / L potassium dihydrogen phosphate, 3 g / L citric acid monohydrate, 3 g / L ammonium sulfate, 5 g / L yeast extract, and 1 mL / L trace elements.

[0163] Glycerol tube inoculation: Take out the glycerol tube from the -80℃ freezer, and use a pipette to inoculate 200μL into LB liquid medium (containing 200mL of medium) in a clean bench. Incubate at 37℃ on a shaker.

[0164] Fermentation tank culture: Inoculate all the cultured bacterial solution into the fermentation tank, containing 9L of fermentation medium.

[0165] The high-density fermentation control conditions are: pH 6.8–7.0, initial temperature 37°C, tank pressure 0.04–0.06 MPa, initial rotation speed 200 rpm, and initial air volume 2–3 L / min.

[0166] After 4-5 hours of fermentation, the dissolved oxygen (DO) suddenly and rapidly rises to over 60%, indicating that the base sugar has been depleted. Feeding is then initiated using a dissolved oxygen feedback feeding method, with dissolved oxygen controlled at 30-55%.

[0167] During fermentation, samples were taken every 2 hours to measure the OD600 value. When the OD reached 20, the inducing agent IPTG was added all at once, with a final concentration of 0.3mM. At the same time, the temperature was lowered to 25℃. After the inducing agent was added, the rotation speed and air volume remained unchanged.

[0168] Fermentation ended after 12 hours of induction, with OD=90. The tank temperature was first lowered to below 20℃, and then the cells were separated by centrifugation.

[0169] Bacterial cells were collected and subjected to protein gel electrophoresis to compare the soluble protein expression after fermentation. Results are shown below. Figure 5 Comparing soluble expression, TEV-M3 transformation of BL21(ΔaldA) yielded the highest proportion of soluble protein (60%).

[0170] Example 5. Optimization of Fermentation Conditions

[0171] High-density fermentation conditions were optimized using different induced OD values, different inducer concentrations, and different temperatures as parameters.

[0172] Glycerol tube inoculation: Take out the glycerol tube from the -80℃ freezer, and use a pipette to inoculate 200μL into LB liquid medium (containing 200mL of medium) in a clean bench. Incubate at 37℃ on a shaker.

[0173] 15L fermenter culture: Inoculate all the cultured bacterial solution into the fermenter, containing 9L of fermentation medium.

[0174] The high-density fermentation control conditions are: pH 6.8–7.0, initial temperature 37°C, tank pressure 0.04–0.06 MPa, initial rotation speed 200 rpm, and initial air volume 2–3 L / min.

[0175] After 4-5 hours of fermentation, the dissolved oxygen (DO) suddenly and rapidly rises to over 60%, indicating that the base sugar has been depleted. At this point, a feedstock (glucose solution containing 700g / L of glucose) is added. The feedstock is a dissolved oxygen feedback type, and the dissolved oxygen is controlled at 30-55%.

[0176] During fermentation, samples were taken every 2 hours to measure the OD600 value. When the OD reached 19-49, the inducing agent IPTG was added all at once, with a final concentration of 0.1-0.5mM. At the same time, the temperature was lowered to 25-30℃. After adding the inducing agent, the rotation speed and air volume remained unchanged.

[0177] Fermentation ended after 12 hours of induction, with an OD of 127-150. The tank temperature was lowered to below 20°C, and then the cells were separated by centrifugation, with a wet weight of 93-128 g / L.

[0178] Take 2g of bacterial cells and perform parallel purification on a smaller volume to compare the concentration after purification. The specific procedure is as follows: Prepare buffer A: 50mM Tris-HCl (pH 8.0), 300mM NaCl, 1mM DTT, 10mM imidazole. Prepare buffer B: 50mM Tris-HCl (pH 8.0), 300mM NaCl, 1mM DTT, 500mM imidazole. Use a protein purification instrument to load the sample, equilibrate, wash with 50mL of 50mM imidazole, and elute with 20mL of 500mM imidazole. Confirm the correct target protein was collected by gel electrophoresis, dialyze, and test the protein concentration.

[0179] The fermentation medium consists of 2 g / L magnesium sulfate heptahydrate, 10 g / L dipotassium hydrogen phosphate, 5 g / L potassium dihydrogen phosphate, 3 g / L citric acid monohydrate, 3 g / L ammonium sulfate, 5 g / L yeast extract, and 1 mL / L trace elements.

[0180] The fermentation results are summarized in Table 9:

[0181] Table 9. Statistical analysis of TEV expression results under different fermentation conditions

[0182]

[0183] Enzyme digestion activity assay for protein product number 2:

[0184] Take 2g of the bacterial cells collected from fermentation No. 2 and perform enzyme activity testing on the purified protein (3.88mg / mL).

[0185] A 750 μL reaction system containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 2 mM MTT, and 1 mM EDTA was prepared, and 15 μM of the experimental substrate SUMO-ENLYFQG-A007 was added. Finally, 0.5 μM TEV was added. A 1 mL reaction system was prepared and reacted at 30 °C and 200 rpm. Samples were taken at 0 h, 1 h, 2 h, 4 h, and 6 h (for gel loading, 20 μL sample + 5 μL of 5x Loading buffer). The enzyme digestion effect was verified. The results are as follows... Figure 6 The results showed that the digested product could be obtained after 1 hour of enzyme digestion, and the digestion was complete after 4 hours.

[0186] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of the cysteine ​​protease of tobacco etch virus, characterized in that, The amino acid sequence of the tobacco etch virus cysteine ​​protease is shown in SEQ ID NO:1, and the mutation site is: T17S, L56V, N68D, I77V, S135G, M121P, W143N, and A195K; Or T17S, L56V, N68D, I77V, S135G, M121P, W143N, A195K and L210N; Or T17S, L56V, N68D, I77V, S135G, W143N, A195K and M218A.

2. The nucleic acid encoding the mutant of claim 1.

3. The nucleic acid according to claim 2, characterized in that, Its nucleic acid sequence is shown in any one of SEQ ID NO:2~4.

4. An expression unit comprising a promoter and the nucleic acid as described in claim 2 or 3.

5. A plasmid vector comprising the nucleic acid of claim 2 or 3 or the expression unit of claim 4.

6. The plasmid vector according to claim 5, characterized in that, Its skeletal carrier is pET28a.

7. A transformant, which is transformed or transfected with the plasmid vector of claim 5 or 6, or whose genome integrates the nucleic acid of claim 2 or 3 or the expression unit of claim 4.

8. The transformant according to claim 7, characterized in that, Its host is Escherichia coli.

9. The transformant according to claim 8, characterized in that, The aldA gene in the E. coli was knocked out.

10. The transformant according to claim 8 or 9, characterized in that, The Escherichia coli strain is either Escherichia coli BL21(DE3) or Escherichia coli Rosetta.

11. The method for preparing the mutant according to claim 1, comprising: The transformant according to any one of claims 7 to 10 is cultured, and the expression of the mutant is induced.

12. The preparation method according to claim 11, characterized in that, The culture medium includes water and magnesium sulfate heptahydrate 2 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 5 g / L, citric acid monohydrate 3 g / L, ammonium sulfate 3 g / L, yeast extract 5 g / L, and trace elements 1 mL / L.

13. The preparation method according to claim 11 or 12, characterized in that, The cultivation conditions include: The pH is 6.8–7.0, the temperature is 37℃, the pressure is 0.04–0.06 MPa, the rotation speed is 200 rpm, the air flow rate is 2–3 L / min, and after culturing for 4–5 hours, the dissolved oxygen is controlled at 30%–55% by fed-batch feeding, and the culture is continued until the OD reaches 6.8–7.

0. 600 The concentration was 18-52, and IPTG was added to a final concentration of 0.1-0.5 mM. The temperature was then lowered to 25-30℃ for 12 h of induction.

14. The preparation method according to claim 13, characterized in that, Cultured to OD 600 The temperature was 28~32℃. IPTG was added to a final concentration of 0.3mM, and the temperature was lowered to 28℃ for 12h induction.

15. The use of the mutant of claim 1, the nucleic acid of claim 2 or 3, the expression unit of claim 4, the plasmid vector of claim 5 or 6, the transformant of any one of claims 7 to 10, or the product obtained by the preparation method of any one of claims 11 to 14, in the preparation of protease preparations.

16. The application according to claim 15, characterized in that, The recognition sequence of the protease is: ENLYFQG.

17. A protease preparation comprising the mutant of claim 1 or the product prepared by any one of claims 11 to 14.

18. A method for protease digestion, comprising treating a protein with the protease preparation of claim 17, wherein the protein is a protein containing the ENLYFQG fragment.

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