TEV protease mutant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to a TEV protease mutant as well as a preparation method and application thereof. According to the invention, TEV enzyme molecules are modified, and mutation sites are different from those reported in the prior art. And the activity of the TEV protease is improved from the molecular level. Moreover, a means of gene editing CRISPR / cas9 is applied to modify a host bacteria chassis, so that an engineering strain for high-yield expression of TEV is obtained. Furthermore, the expression of TEV enzyme can be quickly promoted by a provided high-density fermentation control method, the yield of escherichia coli thalli is high, the OD600 value can reach about 150, the wet weight of the thalli reaches 80-130 g / L, the expression level of target protein is high, and the level of the target protein accounting for the total protein of the thalli can reach 40%-64%. Meanwhile, the method provided by the invention is simple and easy to implement, the used raw materials are simple and easy to obtain, the production cost is reduced while a good application effect is achieved, and remarkable economic benefits are achieved.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a TEV protease mutant and a preparation method and application thereof. Background Art

[0002] TEV enzyme (Tobacco Etch Virus Protease) is the Nla protease derived from tobacco etch virus. It has strong site specificity, and its specific recognition sequence is E-Xaa-Xaa-Y-Xaa-QG / S. The most commonly used sequence is 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. The sequence specificity of TEV enzyme is much higher than that of proteases such as thrombin, factor Xa, and enterokinase. At the same time, the enzyme has a wide range of reaction conditions, and has catalytic activity in the range of reaction pH 5.5-8.5 and reaction temperature 4-30°C.

[0003] It is precisely because of these excellent properties of TEV enzyme that it has been widely used in the field of protein research. Whether it is the in-depth analysis of protein structure or the exploration and excavation of protein function, TEV enzyme plays an indispensable and important role. It has become a powerful tool in the hands of many scientific researchers and continues to contribute to the continuous development of protein research.

[0004] However, the wild-type TEV enzyme inevitably has many defects in its expression and purification process in Escherichia coli. First, the intramolecular self-cleavage phenomenon is relatively serious, which will lead to the instability of the enzyme molecular structure and abnormal changes in function, making it difficult to accurately play a role in subsequent applications. Secondly, a large number of wild-type TEV enzymes exist in the form of inclusion bodies, which greatly reduces the effective content in its active state, and the treatment and conversion of inclusion bodies into active enzyme molecules require complex and cumbersome process steps, which increases production costs and time costs. Furthermore, its output is quite limited and cannot meet the needs of large-scale scientific research and industrial production. In practical applications, it often faces the dilemma of insufficient supply. In addition, the water solubility of TEV enzyme is low, which greatly limits its application in various reaction systems, because many reactions need to be carried out in an aqueous solution environment, and low water solubility will affect the full contact and effective reaction between the enzyme and the substrate.

[0005] Taken together, these defects strictly limit the application scope of wild-type TEV enzyme from multiple dimensions, making it difficult to be effectively used in a wider range of fields and more diverse experimental and production scenarios. In order to solve these problems, some existing technologies help TEV enzyme to fold correctly and achieve soluble expression by selecting a suitable leader peptide, and some improve its preparation method. However, although these methods have solved the problems of low water solubility, poor activity and stability of TEV enzyme expression to a certain extent, they have not solved the problem of large-scale production of TEV. The specific shortcomings are that there has been insufficient research on the expression host strain and how to produce TEV enzyme through high-density fermentation. 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 and a preparation method and application thereof.

[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 comprises 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 the present invention, the cysteine ​​protease shown in SEQ ID NO: 1 is from tobacco etch virus. The present invention uses the TEV mutant of tobacco etch virus cysteine ​​protease that has been published (denoted as M0, containing mutation sites T17S, L56V, N68D, I77V and S135G) and then performs molecular modification.

[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] or including T17S, L56V, N68D, I77V, S135G and A195K;

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

[0016] or including 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] Or include T17S, L56V, N68D, I77V, S135G, A195K and L210N.

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

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

[0051] The present invention optimizes the TEV protease (M0) gene sequence according to the Escherichia coli codon, and further transforms it on this basis to obtain a nucleic acid sequence encoding the mutant of the present 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, in the present invention, the sequence of the nucleic acid is shown in any one of SEQ ID NOs: 2 to 6;

[0053] or a sequence having one or more nucleotides substituted, deleted, added and / or replaced based on the nucleic acid sequence shown in any one of SEQ ID NOs: 2 to 6;

[0054] Or a sequence having a homology of more than 80% with the nucleic acid sequence shown in any one of SEQ ID NOs: 2 to 6.

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

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

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

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

[0059] As mentioned above, the vector containing the nucleic acid or the expression unit is a cloning vector, an expression vector or a viral vector. In the present invention, the vector is used for the storage, amplification or expression of the nucleic acid or the expression unit, and the present invention does not limit this. 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, the fusion protein is expressed using a pET series plasmid vector. As a feasible case, the pET series expression vector is 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, which is transformed or transfected with the plasmid vector as described above, or the nucleic acid as described above or the expression unit as described above is integrated into its genome.

[0061] In the present invention, the host is a eukaryotic host or a prokaryotic host, the eukaryotic host includes but is not limited to yeast, insect cells, kidney epithelial cells, and the prokaryotic host includes but is not limited to Escherichia coli. In some embodiments, the host of the transformant of the present invention is Escherichia coli.

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

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

[0064] In some specific embodiments, the Escherichia coli is an Escherichia coli BL21 (DE3) strain or an Escherichia coli Rosetta strain. More specifically, the Escherichia coli is a BL21 (DE3) strain in which aldA and / or cspD genes are knocked out, or a Rosetta strain in which aldA and / or cspD genes are 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 includes water and 1-5 g / L magnesium sulfate heptahydrate, 5-15 g / L dipotassium hydrogen phosphate, 1-10 g / L potassium dihydrogen phosphate, 1-5 g / L citric acid monohydrate, 1-5 g / L ammonium sulfate, 1-10 g / L yeast extract, and 0.1-1 mL / L trace elements.

[0067] Specifically, the culture medium includes water and 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 powder, and 1 mL / L trace elements.

[0068] The trace elements are mother solutions of trace elements, and each liter of the mother solution of trace elements 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. (calculated per L).

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

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

[0071] More specifically,

[0072] The pH value of the culture is 6.8, 6.9 or 7.0. The initial temperature of the culture 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 0.04MPa, 0.05MPa or 0.06MPa.

[0074] The culture rotation speed is 100-300 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 speed of the culture is 2L / min, 2.2L / min, 2.5L / min, 2.8L / min, 2.9L / min, and 3.0L / min.

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

[0077] The culture is performed to an OD600 of 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 induction is carried out by lowering the temperature to 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0080] The induction duration is 8 to 16 hours, for example, induction for 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 cultured until OD600 is 28-32, IPTG is added to a final concentration of 0.3 mM, and the temperature is lowered to 28° C. for induction for 12 h.

[0082] Furthermore, the present invention also provides the use of the mutant, 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 in the preparation of protease preparations.

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

[0084] Furthermore, the present invention also provides a protease preparation, which includes the mutant, 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 of the present invention also includes a buffer required for enzymolysis. The present invention does not limit the components of the buffer. For example, the buffer is Tris-HCl buffer, HEPES buffer, phosphate buffered saline (PBS).

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

[0087] In the present invention, the enzyme cleavage reaction system includes the protein to be cleaved and 50mM Tris-HCl (pH8.0), 50mM NaCl, 2mM DTT, and 1mM EDTA.

[0088] The present invention has modified the TEV enzyme molecule, and the mutation point is different from that reported previously. The activity of TEV protease is improved at the molecular level. In addition, the present invention uses the gene editing CRISPR / cas9 method to modify the host bacterial chassis to obtain an engineered strain that expresses TEV in high yield. Furthermore, the high-density fermentation control method provided can quickly promote the expression of TEV enzyme, the Escherichia coli cell yield is high, the OD600 value can reach about 150, the cell wet weight can reach 80-130g / L, the target protein expression level is high, and the target protein accounts for 40% to 64% of the total cell protein. At the same time, the method provided by the present invention is simple and easy to implement, the raw materials used are simple and easy to obtain, and while having good application effects, it reduces production costs and has significant economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 . Enzyme cleavage activity results: substrate molecular weight: 65.9 kDa (upper band), cleavage product: 52.3 kDa (lower band), activity is calculated based on the proportion of small molecular weight products generated by cleavage, including: (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 the expression of TEV-M3 in different engineered strains, where: A: host bacteria are BL21(DE3), B: Rosetta, C: BL21(ΔaldA), D: BL21(ΔcspD);

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

[0094] Figure 6 . The enzyme digestion results of TEV-BL21 (ΔaldA) enzyme were obtained in fermentation 2. DETAILED DESCRIPTION

[0095] The present invention provides TEV protease mutants and preparation methods and applications thereof. A person skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0096] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0097] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0098] In this application, "include", "comprising" and "having" are used interchangeably, and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements besides the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions in this article also provides a "consisting of..." solution.

[0099] In the present application, “and / or” when used herein includes the meanings of “and”, “or” and “all or any other combinations of elements linked by the corresponding terms”.

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

[0101] The TEV protease described in the present invention is a cysteine ​​protease derived from tobacco etch virus (TEV). A mutant containing 5 mutation points is used as a starting point, which is numbered as TEV-M0 in this article. It is used as a template for modification. The following is the specific mutation site information (TEV-M1; TEV-M2; M3, M4, M5). The sites after mutation are marked as underlined.

[0102] Table 1 Amino acid sequences of enzymes

[0103]

[0104]

[0105] Table 2 Enzyme encoding nucleic acid sequences

[0106]

[0107]

[0108]

[0109]

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

[0111] The present invention will be further described below in conjunction with embodiments:

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

[0113] After the TEV protease gene sequence was optimized according to the Escherichia coli codon, it was directly synthesized by GenScript Biotech Co., Ltd.; it was inserted into the Escherichia coli expression vector PET-28a plasmid through NdeⅠ and HindⅢ to obtain the PET-28a-7His-TEV expression plasmid, and 5 μL of the expression plasmid was transformed into BL21 (DE3) competent cells, allowed to stand on ice for 30 minutes, then heat shocked at 42°C for 1 minute, and then allowed to stand on ice for 3 minutes, 500 μl of antibiotic-free LB liquid culture medium was added, and the cells were cultured on a shaking incubator at 37°C and 200 rpm for 60 minutes. The cells were spread on an LB plate containing 50 μg / mL kanamycin and cultured in a 37°C incubator overnight to obtain the expression strain.

[0114] Example 2. Mutation construction and screening

[0115] A TEV mutant published in the literature was used as a comparative example, TEV = -M0, containing five mutation points: T17S, L56V, N68D, I77V, S135G). Molecular modification was performed on this parent to obtain a protease with higher properties. TEV-M0 enzyme was used as the parent for rational design assisted by artificial intelligence. The Evcouplings algorithm (protein mutation fitness prediction) was used to calculate the effect of single-point mutations at the evolutionary level, and the ThermoMPNN algorithm (protein thermal stability mutation prediction) was used to calculate the mutation effect on the thermal stability of the TEV enzyme (PDB 1LVM) structure. The prediction results of the two algorithms were weighted and integrated after normalization and sorted. The top 20 mutants with the highest scores were selected, and obviously unreasonable predictions were removed based on experience, such as those that destroyed the original hydrogen bond network and changed active residues. Mutations that could fill structural cavities and replace surface hydrophobic residues with hydrophilic residues were preferred, and 5 rationally designed mutants were screened out: W143N, M121P, A195K, L210N, and M218A. Then, these five mutation points were superimposed, and the AlphaFold2 structure prediction algorithm was used to generate a complex structure model of the superimposed mutants and the substrate ENLYFQG. The structures with the highest pLDDT and ipTM scores were selected, and Gromacs was used for energy optimization and MM / GBSA affinity prediction. The results showed that the affinity of the five superimposed mutants was better than that of the TEV-M0 enzyme in this patent. The five combinations with the highest scores were selected for molecular construction and activity experimental screening. 2.1 Mutant construction:

[0116] Table 3. Primer list

[0117]

[0118]

[0119] Table 4. List of constructed fragments

[0120]

[0121] 2.1.1 PCR cloning and construction of mutants

[0122] PCR reaction system:

[0123] The reaction system was configured according to the following formula:

[0124] Table 5

[0125] primerPF(10uM) 1ul primerPR(10uM) 1ul Template(A0A6P1) 0.5ul(20ng / ul) 5XQ5reactionBuffer 5ul 10mM dNTP 0.5ul Q5polymerase 0.3ul <![CDATA[ddH2O]]> up to 25ul

[0126] PCR machine system:

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

[0128] Table 6

[0129]

[0130] After PCR is completed, the machine is taken off and the PCR products are run on gel electrophoresis. The PCR-positive products are recovered and purified by gel recovery.

[0131] Homologous recombination:

[0132] Recombining and fusing each mutant gene fragment:

[0133] Table 7

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

[0135] Reaction conditions: 50℃ for 30min

[0136] 2.1.2 Transformation: Transform into BL21 (DE3) for expression and preservation.

[0137] Take the homologous recombination product and transfer it into 40ul BL21(DE3) competent cells. After ice bathing for 30min, heat shock at 42℃ for 45s, ice bath for 2-3min, add 500ul SOC, culture on a shaking table for 1.5 hours, centrifuge the whole plate, and culture in a 37℃ incubator overnight for 12-16 hours. Select clones with high expression and store them in a -80℃ refrigerator.

[0138] 2.2 Mutant activity screening:

[0139] 2.2.1 Shake flask culture: Take the seed bacterial solution from the -80 degree refrigerator and streak the plate. The next day, pick the colony from the streaked plate and culture it with 15mL LB medium (50mg / ml Kan antibiotic) at 37℃ and 220rpm. After 5 hours, transfer it to a shake flask with 600mL LB medium and continue to culture. When OD600 reaches 0.8, cool it to 25℃, add 0.3mM IPTG for induction, culture overnight for 15 hours, and collect the bacteria by centrifugation.

[0140] 2.2.2 Purification: 7 protein samples, SUMO-ENLYFQG-A007, TEV-M0, TEV-M1, TEV-M2, M3, M4, M5

[0141] Take 2g of bacteria, crush them by high pressure homogenization, 800bar, 5min. Purify by Ni column, the specific process is: prepare buffer reagent A: 50mM Tris-HCl (pH8.0), 300mM NaCl, 1mM DTT, 10mM imidazole. Reagent B: 50mM Tris-HCl (pH8.0), 300mM NaCl, 1mM DTT, 500mM imidazole. Protein purifier, load, balance, wash (50mM imidazole), elute (500mM imidazole). Confirm that the correct target protein is collected by running gel, dialyze, and store at 4℃ for future use.

[0142] Test protein concentration:

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

[0144] 2.2.3 Test protein activity:

[0145] Add 250 μL of the experimental substrate SUMO-ENLYFQG-A007 to a 750 μL reaction system: 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 2 mM DTT, 1 mM EDTA, and the final concentration is 15 μM. Before the reaction, add different volumes of mutant TEV, and the final concentration is controlled at 0.5 μM. The above 1 mL reaction system is reacted at 30°C and 200 rpm, and samples are taken at 0h (hour), 1h, 2h, 4h, and 6h. (Glue, 20 μL sample + 5 μL 5x Loading buffer.)

[0146] The cleavage activity of different TEV mutants was tested using the SUMO-TEV recognition sequence-protein A (A007) structure: SUMO-ENLYFQG-A007 as a substrate. A mutant that has been published in the literature was used as a comparative example, labeled as TEV-M0. Substrate molecular weight: 65.9 kDa (upper band), cleavage product (A007): 52.3 kDa (lower band). The results are shown in Figure 1 , the statistical results are shown in Table 8:

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

[0148]

[0149] Example 3: Construction of knockout strains

[0150] In order to increase the expression of heterologous proteins, the aldehyde dehydrogenase A gene aldA and the cold shock protein-like protein (DNA replication inhibitor) gene cspD of Escherichia coli BL21 (DE3) were knocked out. By knocking out some unnecessary metabolic pathways in the chassis cells to reduce unnecessary metabolic consumption and increase DNA replication, the expression of the exogenous target protein was increased.

[0151] Build process:

[0152] First, construct sgRNA and donor plasmids: Use the website: http: / / www.rgenome.net / cas-designer / to design sgRNA. Then amplify the backbone, eliminate the plasmid with DpnI, treat with SEAMLESS ligase, and transform DH5a; pick the monoclonal bacteria P for verification, and keep the correct clones. Expand the correct clones and culture them, and save the plasmids for later use. Then, find the aldA and cspD genes on the BL21 (DE3) genome, and select 500bp upstream and downstream as donors; directly use BL21 (DE3) bacteria as templates, amplify 500bp upstream and downstream, and purify by column; fuse the upstream and downstream donors by OEPCR, recover the gel, and save the plasmids for later use.

[0153] Then, pGRB-aldA, cspD plasmids were used to transform BL21 (DE3), specifically: streaking the BL21 (DE3) bacterial solution containing the pECcas9 plasmid on an LB (kan) plate; then, a single clone was picked and cultured overnight in 5 ml LB (kan);

[0154] Transfer 500 μl of the bacterial solution into 50 ml LB (kan), culture for 1-2 hours, adjust the OD to 0.1, add arabinose to a final concentration of 10 mM (1M arabinose, add 500 μl to 50 ml); culture for another 1-2 hours, adjust the OD to about 0.5-0.6, and start preparing the competent state; adjust the refrigerated centrifuge to 4°C for precooling, sterilize 10% glycerol and sterile water and place them in a 4°C refrigerator for precooling, divide the bacterial solution into sterile centrifuge tubes, centrifuge at 6000 rpm for 3 minutes to collect the bacteria; use 40 ml of sterile water washing solution to suspend the cells in each tube, and centrifuge at 6000 rpm for 3 minutes to collect the bacteria; discard the supernatant, use 40 ml of 10% glycerol washing solution to suspend the cells in each tube, and centrifuge at 6000 rpm for 3 minutes to collect the bacteria;

[0155] Resuspend each tube with 3ml 10% glycerol and dispense 100μl per tube into 1.5ml EP tubes; add 100ng pGRB-gRNA plasmid and 200ng donor DNA to 100μl competent cells, electroporate in a pre-cooled 1mm electroporation cup (1.85kV), and immediately suspend in 1ml fresh LB medium after the electroporation. After the cells are incubated at 37°C for 2 hours, all cells are spread on LB plates containing kanamycin (50μg / ml) and ampicillin (50μg / ml) at 37°C overnight. The next day, pick a monoclonal bacterial P for verification (1000bp for successful knockout) (wild type as control), and send the bands that match for testing. Finally, the strain with successful knockout was transferred to 500μl (kan, final concentration 10mM rhamnose (1M rhamnose, 5μl was added to 500μl bacterial solution)) LB and cultured overnight; a loopful of bacterial solution was streaked on the LB (kan) plate. Single clones were picked and streaked on LB (kan) and LB (amp) respectively; the growth on the plates was compared, and those that grew on kan but not on amp should be correct, and the pGRB plasmid had been lost. The strain that lost the pGRB plasmid was inoculated into 2ml LB (antibiotic-free) and cultured overnight; a loopful of bacterial solution was streaked on the LB (sucrose, 10g / L) plate; single clones were picked and streaked on the LB (antibiotic-free) and LB (kan) plates respectively; the growth on the plates was observed, and those that grew on the antibiotic-free plate but not on the kan plate should be strains that lost the pECcas9 plasmid; the strain that successfully lost the plasmid was expanded and cultured (LB, antibiotic-free) for seed preservation. The verification results of successful knockout of genes aldA and cspD are shown in Figure 2 , 3.

[0156] Example 4.

[0157] Transition to different host strains for expression and 15L fermentation screening: conventional BL21(DE3), Rosetta, aldA knockout, cspD knockout for comparison

[0158] 4.1 Comparative experiment of clone expression amount in different strains (4 types):

[0159] In order to further screen out the chassis strain with good expression, the TEV-M3 plasmid was transformed into different host bacteria and the expression was compared: TEV-M3 was expressed in BL21 (DE3) and Rosetta, and 10 clones were selected for each transformation experiment. TEV was transformed into BL21 (ΔaldA) and BL21 (ΔcspD), and the clones on the transformed BL21 (ΔaldA) plate grew well. Five single clones were selected for culture and protein expression, and the expression level was excellent; the BL21 (ΔcspD) plate did not grow normally, and no large number of single clones appeared. Three were selected for culture and expression, and the results on the gel map were not expressed.

[0160] 4.215L fermenter ferments 4 types of strains, expression solubility comparison experiment:

[0161] The four types of engineered strains were screened by fermentation in fermenter.

[0162] The fermentation medium is 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 powder, and 1 mL / L trace elements.

[0163] Glycerol tube inoculation: Take out the glycerol tube from the -80℃ refrigerator, use a pipette to take 200μL and inoculate it into LB liquid medium (containing 200mL of medium) in the clean bench, and culture it in a shaking incubator at 37℃;

[0164] Fermentation tank culture: All the cultured bacterial liquid was inoculated into the fermentation tank, containing 9L of fermentation medium.

[0165] The high-density fermentation control conditions are as follows: 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, DO suddenly and rapidly rebounded to more than 60%, indicating that the base sugar was consumed, and feeding began. The feeding method was dissolved oxygen feedback feeding, and the dissolved oxygen was controlled at 30-55%.

[0167] During the fermentation process, samples were taken every 2 hours to measure the OD600 value. When the OD reached 20, the inducer IPTG was added at a time with a final concentration of 0.3 mM. At the same time, the temperature began to drop to 25°C. After the addition of the inducer, the rotation speed and air volume remained unchanged.

[0168] After 12 hours of induction, the fermentation ended, OD = 90, the tank temperature was first lowered to below 20°C, and then centrifuged to obtain the bacteria.

[0169] Take the bacteria, pass through protein gel, and compare the soluble expression of the protein after fermentation. Figure 5 Comparing the soluble expression, TEV-M3 transformed BL21(ΔaldA), the obtained soluble protein accounted for the highest proportion (60%).

[0170] Example 5. Optimization of fermentation conditions

[0171] The high-density fermentation conditions were optimized with different induction OD, different inducer concentrations, and different temperatures as condition parameters:

[0172] Glycerol tube inoculation: Take out the glycerol tube from the -80℃ refrigerator, use a pipette to take 200μL and inoculate it into LB liquid medium (containing 200mL of medium) in the clean bench, and culture it in a shaking incubator at 37℃;

[0173] 15L fermentation tank culture: All the cultured bacterial liquid was inoculated into the fermentation tank, containing 9L of fermentation medium.

[0174] The high-density fermentation control conditions are as follows: 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, DO (dissolved oxygen) suddenly and rapidly rebounded to more than 60%, indicating that the base sugar was consumed, and feeding (glucose solution, containing 700g / L glucose) was started. The feeding method was dissolved oxygen feedback type feeding, and the dissolved oxygen was controlled at 30-55%.

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

[0177] Fermentation ended after 12 hours of induction, OD = 127-150, the tank temperature was first lowered to below 20°C, and then centrifuged to obtain the bacteria, with a wet weight of 93-128 g / L.

[0178] Take 2g of bacteria and purify in parallel with the same volume of small samples to compare the concentration after purification. The specific process is: prepare buffer reagent A: 50mM Tris-HCl (pH8.0), 300mM NaCl, 1mM DTT, 10mM imidazole. Reagent B: 50mM Tris-HCl (pH8.0), 300mM NaCl, 1mM DTT, 500mM imidazole. Protein purifier, sample loading, balance, wash (50mM imidazole) 50mL, elution (500mM imidazole) 20mL. Confirm that the eluted sample has collected the correct target protein through running gel, dialyze, and test the protein concentration.

[0179] The fermentation medium comprises 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 powder, and 1 mL / L trace elements.

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

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

[0182]

[0183] Enzyme activity detection of protein product No. 2:

[0184] Take 2 g of the bacteria collected from fermentation No. 2 and the purified protein (3.88 mg / mL) for enzyme activity test.

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

[0186] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. 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 comprises at least one of the following mutation sites: T17S, L56V, N68D, I77V, S135G, W143N, M121P, A195K, L210N, M218A.

2. The mutant according to claim 1, characterized in that Mutation sites include T17S, L56V, N68D, I77V, S135G and at least one of the following: W143N, M121P, A195K, L210N, M218A.

3. The mutant according to claim 1, characterized in that the mutation site include: T17S, L56V, N68D, I77V, S135G, M121P, W143N, and A195K; or including T17S, L56V, N68D, I77V, S135G, M121P, W143N, A195K and L210N; or including T17S, L56V, N68D, I77V, S135G, W143N, A195K and M218A; or including T17S, L56V, N68D, I77V, S135G, M121P, W143N, A195K, L210N; and M218A; Or include T17S, L56V, N68D, I77V, S135G, A195K and L210N.

4. A nucleic acid encoding the mutant according to any one of claims 1 to 3.

5. The nucleic acid according to claim 4, characterized in that Its nucleic acid sequence is shown in any one of SEQ ID NOs: 2 to 6; or a sequence having one or more nucleotides substituted, deleted, added and / or replaced based on the nucleic acid sequence shown in any one of SEQ ID NOs: 2 to 6; Or a sequence having a homology of more than 80% with the nucleic acid sequence shown in any one of SEQ ID NOs: 2 to 6.

6. An expression unit comprising a promoter and the nucleic acid according to claim 4 or 5.

7. A plasmid vector comprising the nucleic acid according to claim 4 or 5 or the expression unit according to claim 6.

8. The plasmid vector according to claim 7, characterized in that Its backbone vector is pET28a.

9. A transformant transformed or transfected with the plasmid vector according to claim 7 or 8, or having the nucleic acid according to claim 4 or 5 or the expression unit according to claim 6 integrated into its genome.

10. The transformant according to claim 9, characterized in that Its host is Escherichia coli.

11. The transformant according to claim 10, characterized in that The aldA and / or cspD genes are knocked out in the Escherichia coli.

12. The transformant according to claim 10 or 11, characterized in that The Escherichia coli is the Escherichia coli BL21 (DE3) strain or the Escherichia coli Rosetta strain.

13. The method for preparing the mutant according to any one of claims 1 to 3, comprising: The transformant according to any one of claims 9 to 13 is cultured to induce expression of the mutant.

14. The preparation method according to claim 13, characterized in that: The culture medium comprises water and 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 powder, and 1 mL / L trace elements.

15. The preparation method according to claim 13 or 14, characterized in that: The culture conditions include: The pH value was 6.8-7.0, the temperature was 37°C, the tank pressure was 0.04-0.06MPa, the rotation speed was 200rpm, the air volume was 2-3L / min, and after 4-5h of cultivation, the dissolved oxygen was controlled at 30%-55% by feeding and feeding, and the culture was continued until the OD 600 The cell culture medium was 18-52, IPTG was added to a final concentration of 0.1-0.5 mM, and the temperature was lowered to 25-30°C for induction for 12 h.

16. The preparation method according to claim 15, characterized in that: Culture to OD 600 The temperature was 28-32°C, IPTG was added to a final concentration of 0.3 mM, and the temperature was lowered to 28°C for induction for 12 h.

17. Use of the mutant according to any one of claims 1 to 3, the nucleic acid according to claim 4 or 5, the expression unit according to claim 6, the plasmid vector according to claim 7 or 8, the transformant according to any one of claims 9 to 12, or the product obtained by the preparation method according to any one of claims 13 to 16 in the preparation of a protease preparation.

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

19. A protease preparation comprising the mutant according to any one of claims 1 to 3, the nucleic acid according to claim 4 or 5, the expression unit according to claim 6, the plasmid vector according to claim 7 or 8, the transformant according to any one of claims 9 to 12, or the product obtained by the preparation method according to any one of claims 13 to 16.

20. A method for protease cleavage, comprising treating a protein containing an ENLYFQG fragment with the protease preparation according to claim 19.

Images