High-activity cas enzyme, and preparation method and application thereof

Abstract

This invention relates to a highly active Cas enzyme, its preparation method, and its applications. Specifically, this invention provides a fusion protein containing a CL7 tag, from which high-purity LbCas12a nuclease can be obtained through only two chromatographic steps, including CL7 / Im7 chromatography. The LbCas12a nuclease obtained through the above steps exhibits high activity, stability, and thermostability.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and protein preparation and purification, specifically to a novel high-purity, high-activity Cas enzyme, its preparation method, and its applications. Background Technology

[0002] CRISPR-Cas is a novel nucleic acid detection technology developed in recent years and has been widely used in pathogen detection. Compared with other PCR-based detection technologies, CRISPR / Cas-based nucleic acid detection technologies have the advantages of being convenient, fast, low-cost, and requiring no expensive equipment or professional personnel, making them potential for development into on-site testing tools.

[0003] Cas nucleases are a key component of the CRISPR / Cas system. For proteases, high purity is crucial for ensuring fidelity and efficiency. Traditional Cas nuclease purification methods are cumbersome and time-consuming (>2 days), with each step affecting protein yield and activity. Most importantly, as nucleic acid-binding proteins, Cas proteins readily bind to nucleic acids after bacterial lysis, causing nucleic acid contamination that cannot be completely removed by nickel affinity chromatography. Therefore, conventional purification methods for Cas nuclease preparation often result in low yield, low purity, and poor activity, significantly limiting the development of CRISPR-Cas detection technology. Currently, a novel purification technique, CL7 / Im7 purification technology, has emerged. For nucleic acid-binding proteins, membrane proteins, ultra-large complex proteins, proteins with low expression levels, and proteins difficult to isolate and purify, CL7 / Im7 purification technology achieves high-yield, high-purity, and high-activity preparation.

[0004] In summary, there is an urgent need in this field for a method to prepare high-purity, high-activity, and nuclease-free Cas enzymes using CL7 / Im7 purification technology. Summary of the Invention

[0005] The purpose of this invention is to provide a highly active Cas enzyme, as well as a method for preparing the enzyme and its applications.

[0006] In a first aspect, the present invention provides a fusion protein comprising the structure shown in Formula I:

[0007] Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8(I)

[0008] In the formula, "-" represents a peptide bond or a linking peptide.

[0009] Z1 is a translation enhancement element.

[0010] Z2 is the label sequence.

[0011] Z3 is either empty or has a connector.

[0012] Z4 is an LbCas12a sequence.

[0013] Z5 is either empty or has a connector.

[0014] Z6 is an absent or restricted restriction site.

[0015] Z7 is either empty or has a connector.

[0016] Z8 is an unlabeled or untagged sequence;

[0017] Among them, Z3, Z5, Z6, Z7, and Z8 are not all zero at the same time.

[0018] In another preferred embodiment, the translation enhancement element is an E. coli translation enhancement element.

[0019] In another preferred embodiment, the E. coli translation enhancement element has an amino acid sequence as shown in SEQ ID NO:1.

[0020] In another preferred embodiment, Z2 is a histidine tag.

[0021] In another preferred embodiment, the histidine tag is a 6×His tag.

[0022] In another preferred embodiment, the LbCas12a sequence has an amino acid sequence as shown in SEQ ID NO:3.

[0023] In another preferred embodiment, Z5 is a connector.

[0024] In another preferred embodiment, the linker of Z5 has an amino acid sequence as shown in SEQ ID NO:4.

[0025] In another preferred embodiment, the restriction site is the restriction site of the HRV 3C enzyme.

[0026] In another preferred embodiment, the restriction site has an amino acid sequence as shown in SEQ ID NO:5.

[0027] In another preferred embodiment, Z7 is a connector.

[0028] In another preferred embodiment, the linker of Z7 has an amino acid sequence as shown in SEQ ID NO:6.

[0029] In another preferred embodiment, Z8 is a CL7 tag.

[0030] In another preferred embodiment, the CL7 has an amino acid sequence as shown in SEQ ID NO:7.

[0031] In another preferred embodiment, the fusion protein comprises the structure shown in Formula II:

[0032] Z1-Z2-Z3-Z4-Z5-Z6-Z7(II)

[0033] In the formula, "-" represents a peptide bond or a linking peptide.

[0034] Z1 is a translation enhancement element.

[0035] Z2 is the label sequence.

[0036] Z3 is an LbCas12a sequence.

[0037] Z4 is the connector.

[0038] Z5 is the restriction enzyme site.

[0039] Z6 is the connector.

[0040] Z7 is the tag sequence;

[0041] The translation enhancement element has the amino acid sequence shown in SEQ ID NO:1, the tag sequence of Z2 is a histidine tag, the LbCas12a sequence has the amino acid sequence shown in SEQ ID NO:3, the linker of Z4 has the amino acid sequence shown in SEQ ID NO:4, the restriction enzyme site has the amino acid sequence shown in SEQ ID NO:5, the linker of Z6 has the amino acid sequence shown in SEQ ID NO:6, and Z7 is a CL7 tag.

[0042] In another preferred embodiment, the fusion protein comprises the structure shown in Formula III:

[0043] Z1-Z2-Z3-Z4-Z5(III)

[0044] In the formula, "-" represents a peptide bond or a linking peptide.

[0045] Z1 is a translation enhancement element.

[0046] Z2 is the label sequence.

[0047] Z3 is an LbCas12a sequence.

[0048] Z4 is the connector.

[0049] Z5 is the enzyme cleavage site fragment;

[0050] The translation enhancement element has an amino acid sequence as shown in SEQ ID NO:1, the tag sequence is a histidine tag, the LbCas12a sequence has an amino acid sequence as shown in SEQ ID NO:3, and the Z4 linker has an amino acid sequence as shown in SEQ ID NO:4.

[0051] In another preferred embodiment, the enzyme cleavage site fragment has an amino acid sequence as shown in LEVLFQ (SEQ ID NO:9).

[0052] In another preferred embodiment, the enzyme activity of the fusion protein is significantly increased.

[0053] In another preferred embodiment, "significantly increased" means that, compared with the enzyme activity A0 of the LbCas12a commercial enzyme, the enzyme activity A1 of the fusion protein satisfies A1 / A0≥200% after 5 min of reaction.

[0054] In another preferred embodiment, "significantly increased" means that, compared with the enzyme activity A0 of the commercial LbCas12a enzyme, the enzyme activity A1 of the fusion protein satisfies A1 / A0≥130% after 30 min of reaction.

[0055] In another preferred embodiment, the LbCas12a commercial enzyme comprises: LbCas12a commercial enzyme purchased from NEB.

[0056] In another preferred embodiment, the fusion protein has thermal stability.

[0057] In another preferred embodiment, the thermal stability is obtained by comparing the enzyme activity of the fusion protein stored at -80°C after being placed at room temperature for different times.

[0058] In another preferred embodiment, the enzyme activity H1 of the fusion protein after being placed at room temperature for 24 hours satisfies the condition that H1 / H0 ≥ 95%, compared to the enzyme activity H0 of the fusion protein after being placed at room temperature for 0 hours.

[0059] In another preferred embodiment, the enzyme activity H2 of the fusion protein after being placed at room temperature for 48 hours satisfies the condition that H2 / H0 ≥ 95%, compared to the enzyme activity H0 of the fusion protein after being placed at room temperature for 0 hours.

[0060] In another preferred embodiment, the room temperature is 25°C.

[0061] In another preferred embodiment, the fusion protein is stable.

[0062] In another preferred embodiment, the stability is obtained by comparing the enzyme activity after the fusion protein has been placed at different temperatures for different times.

[0063] In another preferred embodiment, the enzyme activity S1 of the fusion protein stored at -20°C for 5 months satisfies the condition that S1 / S0 ≥ 80%, compared to the enzyme activity S0 of the fusion protein stored at -80°C.

[0064] In another preferred embodiment, the enzyme activity S2 of the fusion protein after two freeze-thaw cycles satisfies the condition that, compared to the enzyme activity S0 of the fusion protein stored at -80°C, S2 / S0 ≥ 80%.

[0065] In a second aspect, the present invention provides a polynucleotide encoding the fusion protein described in the first aspect of the present invention.

[0066] In a third aspect, the present invention provides a carrier comprising the polynucleotide described in the second aspect of the present invention.

[0067] In another preferred embodiment, the vector includes: plasmid vector, viral vector, liposome vector, DNA vector, and RNA vector.

[0068] In another preferred embodiment, the vector is a plasmid vector.

[0069] In another preferred embodiment, the plasmid vector is pCold.

[0070] In another preferred embodiment, the plasmid vector has a nucleotide sequence as shown in SEQ ID NO:8.

[0071] In another preferred embodiment, the plasmid vector includes: a marker gene and an operon.

[0072] In another preferred embodiment, the marker gene includes an ampicillin resistance gene.

[0073] In another preferred embodiment, the ampicillin resistance gene has an amino acid sequence as shown in SEQ ID NO:10.

[0074] In another preferred embodiment, the operon includes a lactose operon.

[0075] In another preferred embodiment, the lactose operon has an amino acid sequence as shown in SEQ ID NO:11.

[0076] In a fourth aspect, the present invention provides a host cell comprising a vector as described in the third aspect of the present invention, or a chromosome incorporating a polynucleotide as described in the second aspect of the present invention, or expressing a fusion protein as described in the first aspect of the present invention.

[0077] In another preferred embodiment, the host cell includes: prokaryotic cells and eukaryotic cells.

[0078] In another preferred embodiment, the prokaryotic cells include Escherichia coli.

[0079] In another preferred embodiment, the host cell does not contain the restriction site of the HRV 3C enzyme.

[0080] In another preferred embodiment, the method for delivering the vector to the host cell includes: thermal shock, electroconversion, chemical conversion, and gene gun method.

[0081] In another preferred embodiment, the method for delivering the vector to the host cell is heat shock.

[0082] In a fifth aspect, the present invention provides an enzyme preparation comprising the fusion protein described in the first aspect of the present invention.

[0083] In another preferred embodiment, the enzyme preparation further includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0084] In another preferred embodiment, the enzyme preparation contains ≥95% of the fusion protein.

[0085] In a sixth aspect, the present invention provides the use of the fusion protein described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the enzyme preparation described in the fifth aspect of the present invention for nucleic acid detection.

[0086] In a seventh aspect, the present invention provides a kit comprising the fusion protein of the first aspect of the present invention, the polynucleotide of the second aspect of the present invention, the vector of the third aspect of the present invention, the host cell of the fourth aspect of the present invention, and the enzyme preparation of the fifth aspect of the present invention.

[0087] In another preferred embodiment, the kit further includes a label or instructions indicating that the kit is used for nucleic acid detection.

[0088] An eighth aspect of the present invention provides a method for preparing the fusion protein described in the first aspect of the present invention, comprising the steps of:

[0089] (1) Under suitable culture conditions, the host cells described in the fourth aspect of the present invention are cultured to obtain the fusion protein described in the first aspect of the present invention.

[0090] In another preferred embodiment, the culture conditions include: culture temperature, culture concentration, culture rotation speed, culture duration, and inducing agent.

[0091] In another preferred embodiment, the culture temperature is 15±1℃, more preferably 15℃.

[0092] In another preferred embodiment, the culture concentration is 0.2 ± 0.02 mM, more preferably 0.2 mM.

[0093] In another preferred embodiment, the culture rotation speed is 220±10 rpm, more preferably 220 rpm.

[0094] In another preferred embodiment, the culture time is 16-18 hours.

[0095] In another preferred embodiment, the inducing agent is IPTG.

[0096] In another preferred embodiment, the method further includes the step of:

[0097] (2) Separate and purify the fusion protein obtained in step (1).

[0098] In another preferred embodiment, the fusion protein is isolated and purified from a culture containing lysed host cells.

[0099] In another preferred embodiment, the method for lysing the host cells includes lysozyme lysis.

[0100] In another preferred embodiment, step (2) includes the following steps:

[0101] (2.1) The unisolated and purified fusion protein was subjected to a first chromatography, and a protease was added to obtain a mixture of the fusion protein and the protease;

[0102] (2.2) Perform a second chromatography on the mixture to obtain the fusion protein.

[0103] In another preferred embodiment, in step (2.1), the first chromatography is performed using a CL7 / Im7 affinity chromatography column.

[0104] In another preferred embodiment, the protease is an HRV 3C enzyme.

[0105] In another preferred embodiment, in step (2.2), the second chromatography is performed using a GST chromatography column.

[0106] In another preferred embodiment, step (2.1) further includes refolding the CL7 / Im7 affinity chromatography column.

[0107] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0108] Figure 1The image shows the results of LbCas12a restriction enzyme digestion verification analysis. M: DL10000 DNA Marker; 1: BamHI and KpnI double-digested recombinant plasmid; 2: KpnI single-digested recombinant plasmid; 3: BamHI single-digested recombinant plasmid; 4: Recombinant plasmid.

[0109] Figure 2 A schematic diagram of the structure of the LbCas12a enzyme is shown.

[0110] Figure 3 The following image shows the SDS-PAGE electrophoresis results of LbCas12a protein purified using the CL7 / Im7 method. A: SDS-PAGE electrophoresis of each protein purification fraction. M: Protein molecular weight standard; 1: Lysis supernatant; 2: Flow-through buffer; 3: High-salt elution buffer; 4: Low-salt elution buffer; 5: Elution buffer 1 after enzyme digestion; 6: Elution buffer 2 after enzyme digestion; 7: Denaturation elution buffer 1; 8: Denaturation elution buffer 2; B: SDS-PAGE electrophoresis of LbCas12a concentration results. M: Protein Marker; 9: Before ultrafiltration concentration (5×); 10: After ultrafiltration concentration; 11: HRV 3C enzyme elution buffer.

[0111] Figure 4 The graph shows the results of the LbCas12a enzyme activity assessment analysis.

[0112] Figure 5 The graph shows the results of the thermostability assessment of the LbCas12a enzyme.

[0113] Figure 6 The graph shows the results of the LbCas12a enzyme stability assessment analysis.

[0114] Figure 7 The results of the LbCas12a enzyme nuclease contamination detection and analysis are shown in the figure. Detailed Implementation

[0115] Through extensive and in-depth research, the inventors have, for the first time, prepared a novel fusion protein that can be used for nucleic acid detection. This fusion protein contains a CL7 tag, and high-purity LbCas12a nuclease can be obtained through only two chromatographic steps, including CL7 / Im7 chromatography. The LbCas12a nuclease obtained through these steps exhibits high activity, stability, and thermostability. Based on this, the present invention was completed.

[0116] It should be understood that the specific methods and experimental conditions of the invention described below in varying degrees of detail are intended to provide a substantive understanding of the invention. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0117] the term

[0118] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the scope of this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0119] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0120] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0121] As used in this article, the terms “CRISPR-Cas system” and “CRISPR / Cas system” are used interchangeably. CRISPR is a nucleic acid detection technology that utilizes Cas nucleases and can also be used in the field of gene editing.

[0122] As used herein, the term "HRV 3C enzyme" refers to the 3C protease of human rhinovirus type 14, also known as PreScissionproteases. This protease specifically recognizes the short peptide Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro at low temperatures and performs enzymatic cleavage between the Gln and Gly amino acid residues. This protease contains a GST tag, allowing it to be separated from mixtures using a GST column.

[0123] CL7 / Im7

[0124] As used herein, the terms “CL7 / Im7”, “CL7 / Im7 method”, “CL7 / Im7 purification method”, “CL7 / Im7 purification technology”, “CL7 / Im7 one-step purification technology”, “CL7 / Im7 one-step purification method”, and “CL7 / Im7 affinity purification technology” are used interchangeably and refer to the method of purifying proteins using the CL7 / Im7 protein purification system.

[0125] This system utilizes a high-affinity complex formed between two small proteins, CL7 and Im7, where CL7 (16 kDa) is the tag for fusion with the target protein and Im7 (10 kDa) is the ligand that couples with agarose resin.

[0126] The CL7 tag can be expressed at the N-terminus or C-terminus of a target protein, increasing the expression level and solubility of proteins in cells (such as E. coli) where the tag is absent. CL7 is a modified bacterial CE7 DNase. After modification, CL7 retains the binding activity of CE7 and Im7 while eliminating its DNase activity.

[0127] Im7 resin consists of Im7 molecules immobilized on agarose resin and serves as the main functional component of Im7 purification columns or CL7 / Im7 columns. Due to the high specific affinity between Im7 and CL7, the off-target binding rate of the resin is extremely low. Im7 resin has a binding capacity of 35-40 mg / mL, thereby capturing a large amount of CL7-tagged proteins.

[0128] Fusion protein

[0129] The fusion protein of this invention is a protein sequence containing the LbCas12a nuclease. Specifically, the fusion protein of this invention comprises the structure shown in Formula I:

[0130] Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8(I)

[0131] In the formula, "-" represents a peptide bond or a linking peptide.

[0132] Z1 is a translation enhancement element.

[0133] Z2 is the label sequence.

[0134] Z3 is either empty or has a connector.

[0135] Z4 is an LbCas12a sequence.

[0136] Z5 is either empty or has a connector.

[0137] Z6 is an absent or restricted restriction site.

[0138] Z7 is either empty or has a connector.

[0139] Z8 is an unlabeled or untagged sequence;

[0140] Among them, Z3, Z5, Z6, Z7, and Z8 are not all zero at the same time.

[0141] Preferably, the translation enhancement element is an E. coli translation enhancement element.

[0142] Preferably, the E. coli translation enhancement element has an amino acid sequence as shown in SEQ ID NO:1.

[0143] Preferably, Z2 is a histidine tag.

[0144] Preferably, the histidine tag is a 6×His tag.

[0145] Preferably, the LbCas12a sequence has the amino acid sequence shown in SEQ ID NO:3.

[0146] Preferably, Z5 is a connector.

[0147] Preferably, the linker of Z5 has an amino acid sequence as shown in SEQ ID NO:4.

[0148] Preferably, the restriction site is the restriction site of the HRV 3C enzyme.

[0149] Preferably, the enzyme cleavage site has an amino acid sequence as shown in SEQ ID NO:5.

[0150] Preferably, Z7 is a connector.

[0151] Preferably, the linker of the Z7 has an amino acid sequence as shown in SEQ ID NO:6.

[0152] Preferably, the Z8 is a CL7 label.

[0153] Preferably, the CL7 has an amino acid sequence as shown in SEQ ID NO:7.

[0154] Preferably, the fusion protein comprises the structure shown in Formula II:

[0155] Z1-Z2-Z3-Z4-Z5-Z6-Z7(II)

[0156] In the formula, "-" represents a peptide bond or a linking peptide.

[0157] Z1 is a translation enhancement element.

[0158] Z2 is the label sequence.

[0159] Z3 is an LbCas12a sequence.

[0160] Z4 is the connector.

[0161] Z5 is the restriction enzyme site.

[0162] Z6 is the connector.

[0163] Z7 is the tag sequence;

[0164] The translation enhancement element has the amino acid sequence shown in SEQ ID NO:1, the tag sequence of Z2 is a histidine tag, the LbCas12a sequence has the amino acid sequence shown in SEQ ID NO:3, the linker of Z4 has the amino acid sequence shown in SEQ ID NO:4, the restriction enzyme site has the amino acid sequence shown in SEQ ID NO:5, the linker of Z6 has the amino acid sequence shown in SEQ ID NO:6, and Z7 is a CL7 tag.

[0165] The fusion protein is purified using the CL7 / Im7 technique to obtain the LbCas12a nuclease required for this invention. Preferably, the fusion protein comprises the structure shown in Formula III:

[0166] Z1-Z2-Z3-Z4-Z5(III)

[0167] In the formula, "-" represents a peptide bond or a linking peptide.

[0168] Z1 is a translation enhancement element.

[0169] Z2 is the label sequence.

[0170] Z3 is an LbCas12a sequence.

[0171] Z4 is the connector.

[0172] Z5 is the enzyme cleavage site fragment;

[0173] The translation enhancement element has an amino acid sequence as shown in SEQ ID NO:1, the tag sequence is a histidine tag, the LbCas12a sequence has an amino acid sequence as shown in SEQ ID NO:3, and the Z4 linker has an amino acid sequence as shown in SEQ ID NO:4.

[0174] Preferably, the enzyme cleavage site fragment has an amino acid sequence as shown in LEVLFQ (SEQ ID NO:9).

[0175] The fusion protein also includes variations of the above sequence. These variations include, but are not limited to: deletions, insertions, and / or substitutions of 1-3 amino acids, and the addition or deletion of one or more amino acids (typically no more than 3, preferably no more than 2, and more preferably no more than 1) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the protein's function. Similarly, the addition or deletion of one or more amino acids at the C-terminus and / or N-terminus typically does not alter the protein's structure and function.

[0176] Polynucleotides

[0177] As used herein, the term "polynucleotide" encoding the fusion protein of the present invention may include the polynucleotide of the fusion protein of the present invention, and may also include polynucleotides of non-coding sequences. The polynucleotides of the present invention also include variants of the above-mentioned polynucleotides that encode polypeptides or fragments, analogs, and derivatives of fusion proteins having the same amino acid sequence as the fusion protein of the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the fusion protein it encodes.

[0178] Vector and host cell

[0179] This invention relates to a vector containing the polynucleotides of the present invention, a host cell containing the vectors of the present invention, and a method for generating the polypeptides of the present invention using recombinant technology.

[0180] Preferably, the vector is an expression vector. Expression vectors well-known in the art include plasmid vectors, bacteriophages, and viral vectors. Specifically, the plasmid vector includes bacterial plasmids and yeast plasmids; the viral vector includes plant viruses and animal viruses (such as adenoviruses and retroviruses). The expression vector used can stably exist and replicate in host cells. Important characteristics of expression vectors also include the presence of an origin of replication, a promoter, a marker gene, and translational control elements.

[0181] Methods for constructing expression vectors containing the coding nucleic acid sequence of the fusion protein of this invention are well known to those skilled in the art. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The promoters include: the lac or trp promoter of *E. coli*; the PL promoter of λ phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0182] The expression vector also includes one or more marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase genes, neomycin resistance genes, and green fluorescent protein (GFP) genes for eukaryotic cell culture, or tetracycline genes or ampicillin resistance genes for Escherichia coli. Preferably, the marker gene is an ampicillin resistance gene.

[0183] Methods for generating the polypeptides described in this invention using recombinant technology are well known to those skilled in the art. These methods typically include the following steps: (1) transforming or transducing suitable host cells with a polynucleotide encoding the fusion protein of this invention, or with a vector containing the polynucleotide; (2) culturing the host cells in a suitable culture medium; and (3) isolating and purifying the protein from the culture.

[0184] Methods for transforming or transducing host cells are well known to those skilled in the art. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of absorbing exogenous DNA can be harvested after the exponential growth phase. Treatment with CaCl2 or MgCl2 is well known in the art. Electroporation can also be used for transformation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

[0185] The transformed host cells can be cultured using conventional methods to express the fusion protein of this invention. The type of culture medium and culture conditions are selected based on the host cells used. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0186] Using the methods described above, fusion proteins can be expressed intracellularly, on the cell membrane, or secreted extracellularly. Recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0187] The main advantages of this invention include:

[0188] (1) The present invention unexpectedly discovered that the LbCas12a nuclease provided by the present invention has the characteristic of high activity. When the reaction is 5 min, the activity of the LbCas12a nuclease is twice that of the commercial LbCas12a nuclease; when the reaction is 30 min, the activity of the LbCas12a nuclease is 1.3 times that of the commercial LbCas12a nuclease.

[0189] (2) The present invention also unexpectedly discovered that the LbCas12a nuclease has thermal stability and stability.

[0190] (3) The LbCas12a nuclease prepared by the preparation method described in this invention has high purity, reaching 95%, and is free from nuclease contamination.

[0191] (4) The LbCas12a nuclease can be prepared by the preparation method described in this invention with fewer steps and higher recovery rate.

[0192] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0193] Example 1: Construction of LbCas12a recombinant plasmid and acquisition of LbCas12a

[0194] 1. Obtaining the LbCas12a gene fragment: Using the pMBP-LbCas12a vector as a template, the LbCas12a gene fragment was amplified by high-fidelity PCR.

[0195] 2. Construction of LbCas12a recombinant plasmid: In this invention, the LbCas12a gene fragment amplified by PCR reaction is cloned into the pCold linearized plasmid using seamless ligation technology to construct the LbCas12a recombinant plasmid.

[0196] The constructed LbCas12a recombinant plasmid was verified by enzyme digestion, and the plasmids that passed the enzyme digestion verification were sequenced. The enzyme digestion verification results are as follows: Figure 1 As shown.

[0197] 3. Transformation of LbCas12a recombinant plasmid into *E. coli*: The LbCas12a recombinant plasmid was transformed into *E. coli* BL21(DE3)plysS competent cells using a heat shock transformation method. The specific procedure was as follows: A tube of 100 μL BL21(DE3)plysS competent cells was taken from a -80°C freezer and thawed on ice. Then, 10 ng of the constructed recombinant plasmid was added and gently mixed. The cells were incubated on ice for 30 minutes. Next, the cells were heat-shocked in a 42°C water bath for 90 seconds, then quickly returned to ice and incubated for 3 minutes. 900 μL of LB liquid medium was added to the tube, and the cells were cultured at 37°C and 220 rpm for 60 minutes to revive the resistance gene. Finally, 100 μL of the 800 μL bacterial culture was taken and evenly spread on a plate containing ampicillin and chloramphenicol resistance markers. The plate was then inverted and incubated overnight at 37°C. The obtained positive clones are recombinant *E. coli* strains containing the LbCas12a recombinant plasmid.

[0198] 4. Induction of expression in the expression bacteria: 2% was inoculated into the corresponding resistant TB liquid medium and incubated at 37℃ and 220 rpm until the optical density (OD) of the bacterial solution was measured. 600 Once the concentration reached 0.6, 0.2 mM IPTG was added, and expression was induced overnight at 15℃ / 220 rpm. The cells were then centrifuged at 4℃ and 10000g for 3 min, and the bacterial pellet was collected.

[0199] 5. Cell lysis: The collected bacterial cells were lysed using the lysozyme lysis method. An appropriate volume of lysis buffer was added to resuspend the cells, followed by the addition of lysozyme, MgCl2, and nuclease to fully lyse the cells. The lysed cells were centrifuged at high speed for 30 minutes, and the supernatant was collected.

[0200] A solution containing the LbCas12a nuclease protein can be obtained by inducing expression and then lysing bacterial cells. The sequence structure of the nuclease is as follows. Figure 2 As shown in Table 1. The amino acid sequence of the nuclease is shown in Table 2. The nucleic acid sequence of the expression vector containing the nuclease is shown in Table 3.

[0201] Table 1. Amino acid sequence of LbCas12a nuclease

[0202]

[0203]

[0204] Table 2. Nucleic acid sequences of the expression vector (SEQ ID NO:8)

[0205]

[0206]

[0207]

[0208] Table 3. Amino acid sequences of marker genes and operators contained in expression vectors

[0209]

[0210] Example 2: Preparation of LbCas12a protein using CL7 / Im7 one-step purification technology

[0211] This embodiment involves the preparation of LbCas12a protein using the CL7 / Im7 purification method. The specific steps are as follows:

[0212] (1) Pass the protein supernatant obtained in Example 1 through a column, and then wash off the impurities with high-salt buffer and low-salt buffer alternately.

[0213] (2) Equilibrate the CL7 / Im7 column with HRV 3C enzyme reaction solution;

[0214] (3) Add 20U HRV 3C enzyme, digest on column at 4℃ for 2h, and collect the flow-through solution after digestion to obtain the LbCas12a protein and HRV 3C enzyme mixture;

[0215] (4) Denaturing buffer was added to the CL7 / Im7 affinity chromatography column to denature it and remove the CL7-tagged peptide;

[0216] (5) Finally, add refolding buffer to slowly refold the CL7 / Im7 column at a 10% gradient;

[0217] (6) Use a GST column to remove the residual HRV 3C enzyme in the flow-through solution after enzyme digestion to obtain high-purity LbCas12a protein.

[0218] The purified LbCas12a protein was concentrated using an ultrafiltration tube with a flow rate of 30 kDa, and the storage buffer was replaced. The concentrated LbCas12a protein was then mixed with an equal volume of glycerol and stored at -80°C for long-term storage.

[0219] Example 3: Identification of the purity of LbCas12a nuclease

[0220] This embodiment involves the identification of the purity of LbCas12a nuclease.

[0221] The protein concentration was determined by BCA method, and the high purity LbCas12a protein with a protein content of 5.73 mg / mL was obtained.

[0222] The LbCas12a protein prepared by the CL7 / Im7 one-step purification technique was loaded onto a sample and subjected to SDS-PAGE gel electrophoresis for purity identification.

[0223] The results are as follows Figure 3 As shown, the LbCas12a protein prepared by the CL7 / Im7 one-step purification technique has a purity greater than 95%, which is high-purity LbCas12a protein.

[0224] Example 4: Evaluation of LbCas12a nuclease activity

[0225] This embodiment relates to the detection of the enzyme activity of LbCas12a nuclease, specifically by evaluating the activity of LbCas12a prepared by CL7 / Im7 purification technology through an in vitro cleavage experiment.

[0226] To eliminate interference from target and crRNA design, the standards and crRNA stored in this experiment were used. The flanking cleavage activity of LbCas12a enzymes from three different sources was tested using 3.5 nM Toxoplasma gondii standards. The three different sources of LbCas12a enzymes were: the LbCas12a enzyme obtained by purification using the CL7 / Im7 method in this invention, the commercially available LbCas12a enzyme from New England Biolabs (NEB), and a domestically produced LbCas12a enzyme.

[0227] The reaction system was configured as shown in Table 4. The reaction was carried out at 37℃ for 30 min using a Suzhou Kerui MA-1610 isothermal amplification instrument. The FAM channel was selected, and the fluorescence value was read every 1 min.

[0228] Table 4. Nuclease Stability Detection System

[0229]

[0230]

[0231] The results are as follows Figure 4 As shown, LbCas12a prepared using the CL7 / Im7 method exhibited the highest enzyme activity, significantly higher than the other two commercially available enzymes.

[0232] Example 5: Evaluation of the thermostability of LbCas12a nuclease

[0233] This embodiment involves evaluating the thermal stability of LbCas12a nuclease, specifically by detecting the thermal stability of LbCas12a prepared by CL7 / Im7 purification technology through an in vitro cleavage experiment.

[0234] Three identical tubes of LbCas12a were used in the experiment, configured as shown in Table 1. The tubes were incubated at room temperature for 0 h, 24 h, and 48 h, respectively. Lateral cleavage activity assays were then performed to assess the thermal stability of LbCas12a, with three replicates per group. The reaction was conducted using a Suzhou Kreek isothermal amplification instrument for 30 min, and fluorescence values ​​were read.

[0235] The results are as follows Figure 5 As shown, LbCas12a prepared by the CL7 / Im7 method exhibits high thermal stability. The fluorescence rate and absolute fluorescence value of the experimental group's LbCas12a enzyme within the same time frame were almost identical to those of the control group, indicating that the LbCas12a enzyme purified by the CL7 / Im7 method has relatively stable activity.

[0236] Example 6: Evaluation of the stability of LbCas12a nuclease

[0237] This embodiment involves evaluating the stability of LbCas12a nuclease, specifically by detecting the stability of LbCas12a prepared by CL7 / Im7 purification technology through an in vitro cleavage experiment.

[0238] To further investigate the stability of LbCas12a protein, the experimental setup included a negative control without LbCas12a, a positive control of LbCas12a protein stored at -80℃, and experimental groups consisting of one tube of protein stored at -20℃ for 5 months and one tube of protein subjected to two freeze-thaw cycles. Each group had three replicates, and the reaction system was configured as shown in Table 1. The stability of LbCas12a was further assessed using an in vitro para-lateral cleavage assay. The reaction was performed for 30 minutes using a Suzhou Kerui isothermal amplification instrument, and fluorescence values ​​were recorded.

[0239] The results are as follows Figure 5 and Figure 6 As shown, the fluorescence rate and absolute fluorescence value of the LbCas12a enzyme in the experimental group were almost identical to those in the control group within the same time period, with no significant decrease. This indicates that the LbCas12a enzyme purified by the CL7 / Im7 method has relatively stable activity, meaning that the self-made LbCas12a enzyme has not been degraded, and the LbCas12a prepared by the CL7 / Im7 method has high stability.

[0240] Example 7: Detection of LbCas12a nuclease contamination

[0241] This embodiment relates to the detection of LbCas12a nuclease contamination level using agarose gel electrophoresis.

[0242] The LbCas12a protein prepared using CL7 / Im7 was tested for nuclease contamination using an in vitro cleavage assay. The reaction system is shown in Table 5. A control group without LbCas12a nuclease was used. The mixture was incubated at 37°C for 60 min and then verified by 1% agarose gel electrophoresis.

[0243] Table 5. Nuclease contamination detection system

[0244]

[0245] The results are as follows Figure 7 As shown, the bands of LbCas12a purified by CL7 / Im7 are almost identical to those of the control group, indicating that LbCas12a purified using this invention is free from nucleic acid and nuclease contamination.

[0246] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises the structure shown in Formula I: Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I) In the formula, "-" represents a peptide bond. Z1 is a translation enhancement element. Z2 is the label sequence. Z3 is LbCas12a. Z4 is the connector. Z5 is an enzyme cleavage site or an enzyme cleavage site fragment. Z6 is either empty or has a connector. Z7 is an unlabeled or tagless sequence; Wherein, Z2 is a histidine tag, the restriction site is the restriction site of HRV 3C enzyme, and the tag sequence of Z7 is the CL7 tag; When Z5 is a restriction enzyme site, Z6 is an adapter and Z7 is a tag sequence; when Z5 is a restriction enzyme site fragment, both Z6 and Z7 are absent. The amino acid sequence of the translation enhancement element is SEQ ID NO:1, the amino acid sequence of LbCas12a is SEQ ID NO:3, the amino acid sequence of the linker of Z4 is SEQ ID NO:4, the amino acid sequence of the restriction enzyme site is SEQ ID NO:5, the amino acid sequence of the restriction enzyme site fragment is LEVLFQ (SEQ ID NO:9), and the amino acid sequence of the linker of Z6 is SEQ ID NO:

6.

2. The fusion protein as described in claim 1, characterized in that, The fusion protein comprises the structure shown in Formula I: Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I) In the formula, "-" represents a peptide bond. Z1 is a translation enhancement element. Z2 is the label sequence. Z3 is LbCas12a. Z4 is the connector. Z5 is the restriction enzyme site. Z6 is the connector. Z7 is the tag sequence; Wherein, the amino acid sequence of the translation enhancement element is SEQ ID NO:1, the tag sequence of Z2 is a histidine tag, the amino acid sequence of LbCas12a is SEQ ID NO:3, the amino acid sequence of the linker of Z4 is SEQ ID NO:4, the amino acid sequence of the restriction enzyme site is SEQ ID NO:5, the amino acid sequence of the linker of Z6 is SEQ ID NO:6, and Z7 is a CL7 tag.

3. The fusion protein as described in claim 1, characterized in that, The fusion protein comprises the structure shown in Formula II: Z1-Z2-Z3-Z4-Z5 (II) In the formula, "-" represents a peptide bond. Z1 is a translation enhancement element. Z2 is the label sequence. Z3 is LbCas12a. Z4 is the connector. Z5 is the enzyme cleavage site fragment; Wherein, the amino acid sequence of the translation enhancement element is SEQ ID NO:1, the tag sequence is a histidine tag, the amino acid sequence of LbCas12a is SEQ ID NO:3, and the amino acid sequence of the Z4 linker is SEQ ID NO:4; The amino acid sequence of the enzyme cleavage site fragment is LEVLFQ (SEQ ID NO:9).

4. The fusion protein as described in claim 3, characterized in that, The enzyme activity of the fusion protein is significantly increased; the "significant increase" means that, compared with the enzyme activity A0 of the commercial LbCas12a enzyme, the enzyme activity A1 of the fusion protein satisfies A1 / A0≥200% at 5 min of reaction and A1 / A0≥130% at 30 min of reaction.

5. The fusion protein as described in claim 3, characterized in that, The fusion protein is thermally stable; compared with the enzyme activity H0 of the fusion protein placed at room temperature for 0 hours, the enzyme activity H1 of the fusion protein placed at room temperature for 24 hours satisfies: H1 / H0≥95%; the enzyme activity H2 of the fusion protein placed at room temperature for 48 hours satisfies: H2 / H0≥95%.

6. The fusion protein as described in claim 3, characterized in that, The fusion protein is stable; compared with the enzyme activity S0 of the fusion protein stored at -80℃, the enzyme activity S1 of the fusion protein stored at -20℃ for 5 months satisfies: S1 / S0≥80%; the enzyme activity S2 of the fusion protein after two freeze-thaw cycles satisfies: S2 / S0≥80%.

7. A polynucleotide, characterized in that, The polynucleotide encodes the fusion protein of claim 1.

8. A carrier, characterized in that, The carrier comprises the polynucleotide of claim 7.

9. A host cell, characterized in that, The host cell includes the vector of claim 8, or a chromosome in which the polynucleotide of claim 7 is integrated, or which expresses the fusion protein of claim 1.

10. An enzyme preparation, characterized in that, The enzyme preparation comprises the fusion protein of claim 3, wherein the fusion protein has the structure shown in Formula II.

11. Use of the fusion protein of claim 1, the polynucleotide of claim 7, the vector of claim 8, the host cell of claim 9, or the enzyme preparation of claim 10, characterized in that, Used for nucleic acid testing.

12. A reagent kit, characterized in that, The kit comprises the fusion protein of claim 1, the polynucleotide of claim 7, the vector of claim 8, the host cell of claim 9, and the enzyme preparation of claim 10.

13. A method for preparing the fusion protein according to claim 1, characterized in that, Including the following steps: (1) The host cell of claim 9 is cultured under suitable culture conditions to obtain the fusion protein of claim 1.

14. The preparation method according to claim 13, characterized in that, The method further includes the following steps: (2) Separate and purify the fusion protein obtained in step (1).

15. The preparation method according to claim 14, wherein step (2) includes the step of: (2.1) The unisolated and purified fusion protein is subjected to a first chromatography, and a protease is added to obtain a mixture of the fusion protein and the protease, wherein the fusion protein in the mixture has the structure shown in Formula II; (2.2) The mixture is subjected to a second chromatography to obtain a fusion protein having the structure shown in Formula II: Z1-Z2-Z3-Z4-Z5 (II), where "-" represents a peptide bond, Z1 is a translational enhancement element, Z2 is a tag sequence, Z3 is LbCas12a, Z4 is a linker, and Z5 is an enzyme cleavage site fragment; wherein... The amino acid sequence of the translation enhancement element is SEQ ID NO:1, the tag sequence is a histidine tag, the amino acid sequence of LbCas12a is SEQ ID NO:3, the amino acid sequence of the Z4 linker is SEQ ID NO:4, and the amino acid sequence of the enzyme cleavage site fragment is LEVLFQ (SEQ ID NO:9).

16. The preparation method according to claim 15, characterized in that, In step (2.1), the first chromatography is performed using a CL7 / Im7 affinity chromatography column.

17. The preparation method according to claim 15, characterized in that, In step (2.2), the second chromatography is performed using a GST chromatography column.

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