High-temperature endonuclease protein designed from de novo as well as preparation method and application of high-temperature endonuclease protein

Abstract

The invention discloses a high-temperature endonuclease protein designed from the beginning as well as a preparation method and application of the high-temperature endonuclease protein. The high-temperature endonuclease protein designed from the beginning refers to an Ago protein which has relatively low sequence similarity with a PfAgo protein and is designed by virtue of AI aided design through a GNN model. Compared with a wild type, the protein disclosed by the invention has the advantages that the thermal denaturation midpoint temperature (Tm) of the protein is obviously reduced, and the high-temperature enzyme catalytic activity is obviously improved and can be up to 10 times to the maximum. The method has application potential in nucleic acid detection technology and gene editing tool development technology.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and relates to a de novo designed high-temperature endonuclease protein and a preparation method and application thereof. Background Art

[0002] Argonaute (Ago) proteins, as an important component of RNA-induced silencing complex (RISC), play a critical role in the shearing and recruitment of small RNA molecules. They are a protein family that has attracted much attention since the discovery of RNA interference (RNAi). It has been found that Argonaute proteins are widely present in eukaryotic and prokaryotic organisms, and their functions in eukaryotic and prokaryotic organisms are different. In eukaryotic organisms, Ago proteins are mainly involved in the RNAi process and play an important role in the formation of the RISC complex; in prokaryotes, Ago proteins mainly play a host defense function, participate in the defense mechanism of bacteria against foreign gene invasion, and participate in the replication and damage repair of the host.

[0003] Prokaryotic Argonaute proteins (pAgos for short) are more diverse in function and structure than eAgos, but their physiological functions have long been elusive. Early studies focused on pAgos from thermophilic organisms. Except for MpAgo, which prefers to use RNA guides (gDNA) with 5' terminal hydroxylation (5'OH) to cut target single-stranded DNA (ssDNA) and target RNA, the remaining pAgos from thermophilic organisms prefer to use gDNA with 5' terminal phosphorylation (5'P) to cut target ssDNA and / or target RNA. pAgos from thermophilic organisms have only low levels of gDNA-guided target ssDNA and / or target RNA cutting activity under high temperature conditions, which limits the application development based on thermophilic pAgos. Recent studies have begun to focus on pAgos from thermophilic organisms to obtain pAgos that can effectively cut target DNA and / or target RNA under high temperature conditions. Similar to the commonly used CRISPR-Cas9 and CRISPR-Cas12a / 13a, some people also suggest that pAgos be used as the next generation of genome editing tools. PfAgo protein is a type of high-temperature endonuclease discovered so far. Its wild type has no activity at 65-75℃ and only has low nuclease activity at 85℃, which greatly limits its application in gene editing and nucleic acid detection.

[0004] Traditional protein engineering techniques such as directed evolution make small perturbations to natural protein sequences. In essence, it is a trial-and-error method. It is inefficient without high-throughput screening and it is difficult to create proteins with new structures and functions. With the rapid development of computer technology and AI, the application of computers in enzyme engineering has continuously expanded the exploration of enzyme sequence space. Based on the needs of structure and function, the method of determining amino acid sequences by computational means can be used for both de novo protein design and the modification and design of existing proteins. It is a development direction that needs to be promoted urgently.

[0005] Therefore, it is urgent to develop a method to obtain PfAgo mutants with low reaction temperature and high catalytic activity. And the modification of PfAgo catalytic activity will further promote the development of protein de novo design technology. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a de novo designed high temperature endonuclease protein and its preparation method and application, so as to improve the enzyme activity and thermal stability.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A de novo designed high-temperature endonuclease protein PfAgo or its active fragment, variant form, or derivative protein, wherein the high-temperature endonuclease protein PfAgo has an amino acid sequence shown in any one of SEQ ID NOs. 1 to 3 (Pf-19, Pf-65, or Pf-71), or an amino acid sequence having at least 50% identity with SEQ ID NOs. 1 to 3; the high-temperature endonuclease is derived from the high-temperature prokaryotic organism Pyrococcus furiosus, and the sequence is designed by AI.

[0009] As one of the preferred technical solutions, the high temperature nuclease protein PfAgo has an amino acid sequence shown in SEQ ID NO.1 or 2, namely Pf-19 or Pf-65.

[0010] As one of the preferred technical solutions, the high temperature is 10-95°C, more preferably 60-95°C, and even more preferably 65°C.

[0011] As one of the preferred technical solutions, the ratio of the enzyme activity Q1 of the PfAgo mutant protein to the enzyme activity Q0 of the wild type: Q1 / Q0 is ≥2, preferably ≥3, and optimally ≥7.

[0012] As one of the further preferred technical solutions, the ratio of the enzyme activity Q1 of the PfAgo mutant protein to the enzyme activity Q0 of the wild type: the Q1 / Q0 ratio is 2.0-3.0, preferably 10.

[0013] As one of the preferred technical solutions, the active fragments, variant forms, and derived proteins include:

[0014] The amino acid sequence formed by the aforementioned single-site mutation on the sequence shown in SEQ ID NO.1-3, further having one or more (for example, usually 1-30, preferably 1-10, more preferably 1-6, more preferably 1-3, most preferably 1) amino acid residues deleted, inserted and / or substituted, still has the activity of directed cleavage of single-stranded DNA (cA1), which is significantly higher than the corresponding activity (cA0) of the wild-type PfAgo enzyme shown in SEQ ID NO.4, wherein the significantly higher value is any value in the range of (cA1-cA0) / cA0≥10%-800%, for example, ≥15%, ≥20%, ≥40%, ≥50%, ≥100%, ≥200%, or ≥600% or higher;

[0015] Modification of proteins (usually without changing the primary structure), including: chemical derivatization of proteins in vivo or in vitro such as acetylation or carboxylation; glycosylation; sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine); proteins modified to improve their resistance to proteolysis or optimize their solubility;

[0016] Fusion proteins or conjugates formed by the aforementioned proteins or their active fragments and other proteins or markers;

[0017] Contains one or more other mutations to further enhance the enzymatic activity of the de novo designed PfAgo protein.

[0018] 2. An isolated polynucleotide encoding the aforementioned de novo designed protein PfAgo or its active fragment, variant form, or derivative protein.

[0019] 3. A vector comprising the isolated polynucleotide described above.

[0020] As one of the preferred technical solutions, the vector is a recombinant expression vector, which refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as they can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0021] As one of the preferred technical solutions, the polynucleotide can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as the nucleotide sequence encoding the PfAgo mutant protein in the embodiment of the present invention or a degenerate variant.

[0022] 4. A host cell containing the aforementioned vector, or a host cell containing the aforementioned isolated polynucleotide in its nucleic acid.

[0023] As one of the preferred technical solutions, the host cell includes cells derived from the following microorganisms:

[0024] Saccharomyces cerevisiae, Pichia pastoris, Saccharo myces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipites, Candida shehatae, Candida tropicalis, Escherichia coli.

[0025] The protein can be designed from scratch by the conventional recombinant transformation method in the art, and the de novo designed protein can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its 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 renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic breaking, ultrasonic treatment, high pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods. The host cell of the present invention can express PfAgo protein mutant protein.

[0026] As one of the further preferred technical solutions, the host cell is cultured under conditions suitable for expression, thereby expressing the PfAgo mutant protein; and

[0027] The PfAgo protein is isolated.

[0028] The obtained mutant protein can also be optionally purified to obtain a purer mutant protein product.

[0029] As one of the further preferred technical solutions, the conditions suitable for expression include conventional techniques in the art, and the purification techniques include nickel column purification, ion exchange chromatography, and the like.

[0030] 5. A method for preparing the aforementioned de novo designed high-temperature nuclease protein PfAgo or its active fragment, variant form, or derivative protein, culturing the aforementioned host cell to express the mutant protein or its active fragment, variant form, or derivative protein.

[0031] 6. Application of the aforementioned de novo designed high-temperature endonuclease protein PfAgo or its active fragments, variant forms, and derivative proteins in nucleic acid detection and gene editing.

[0032] 7. An in vitro DNA cleavage system based on the aforementioned de novo designed protein PfAgo or its active fragments, variant forms, and derivative proteins, comprising:

[0033] (A) a guide DNA (gDNA), wherein the guide DNA (gDNA) binds to a predetermined target site; and

[0034] (B) programmable endonuclease Argonaute (PfAgo), wherein the programmable endonuclease is the aforementioned PfAgo mutant protein or its active fragment, variant form, derivative protein; and

[0035] (C) A target DNA having a nucleotide sequence that is largely complementary to the guide DNA.

[0036] As one of the preferred technical solutions, the length of the guide DNA is 21 to 45 nt, more preferably 15 to 21 nt, and even more preferably 16 to 18 nt.

[0037] As one of the preferred technical solutions, the guide DNA and the target nucleic acid have reverse complementary fragments.

[0038] As one of the preferred technical solutions, the target DNA is a target nucleic acid carrying a reporter molecule, and the reporter molecule is a nucleic acid molecule carrying a fluorescent group and a quenching group respectively.

[0039] As one of the further preferred technical solutions, the fluorescent group includes: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof, and FAM is further preferred.

[0040] As one of the further preferred technical solutions, the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof, and more preferably BHQ.

[0041] 8. A method for cutting DNA in vitro based on the above system, the specific steps are as follows:

[0042] S1. Formation of PfAgo-guide complex based on PfAgo and gDNA;

[0043] S2. The PfAgo-guide complex is brought into contact with the target DNA, and the PfAgo-guide complex cuts the target DNA at a specific site.

[0044] As one of the preferred technical solutions, the nuclease activity of the PfAgo and PfAgo-guide complex requires the presence of at least one selected from Mn 2+ ,Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ ,Co 2+ , Zn 2+ and Ni 2+ Or any combination of divalent metal ions therein.

[0045] As one of the further preferred technical solutions, the cations of PfAgo and PfAgo-guide complex are Mn 2 + .

[0046] As one of the further preferred technical solutions, the concentration of the divalent metal ion is 10 μM to 3 mM, more preferably 10 μM to 200 mM, and even more preferably 20 μM.

[0047] The beneficial effects of the present invention are:

[0048] The present invention has obtained a mutant protein with significantly improved activity of the PfAgo enzyme through extensive and in-depth research and a large number of screenings. It is a multi-point mutant protein designed from scratch based on the high temperature nuclease Argonaute (PfAgo, the wild-type sequence shown in SEQ ID NO.4) of Pyrococcus furiosus or its active fragment, variant form, and derivative protein. The PfAgo de novo designed protein derivative protein sequence is the same as the sequence shown in SEQ ID NO.1-3.

[0049] The present invention uses the GNN model to learn different pAgo protein structural information, and uses Alpahfold and other screening methods to select AI-designed Ago proteins from scratch, and obtains mutants with improved enzyme catalytic activity through screening. In the present invention, a total of 3 highly active mutants were finally selected.

[0050] The PfAgo mutant protein of the present invention has nuclease activity at a temperature range of 10 to 95° C., preferably at a high temperature of 60 to 95° C., and more preferably at 65° C. The enzyme catalytic activity of the PfAgo mutant protein of the present invention is significantly improved compared with the wild type, and has better high temperature thermal stability.

[0051] The PfAgo mutant protein significantly improves the activity of directional shearing of single-stranded DNA targets, with the activity improvement reaching 2-10 times, thus providing assistance for pathogen detection, genotyping, disease course monitoring, etc. The method of the present invention can perform site-specific modification on extracellular genetic material, so it can be effectively applied in biotechnology fields such as nucleic acid detection and gene editing. This increases the application potential of the protein in the field of nucleic acid detection technology, and also lays the foundation for the development of new genetic manipulation tools.

[0052] The main advantages of the present invention are as follows:

[0053] 1. The mutants screened by the present invention have significantly improved enzyme catalytic activity compared with the wild type, up to 10 times;

[0054] 2. The thermal stability of the mutant of the present invention is not adversely affected by the mutation and maintains very good thermal stability;

[0055] 3. The mutants of the present invention have application potential in nucleic acid detection technology and the development of gene editing tool technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for explanation.

[0057] Figure 1The SDS-PAGE electrophoresis diagram of wild-type PfAgo and mutants, where M: Marker; Lane 1: purified WT sample; Lanes 2-4: purified mutant protein samples;

[0058] Figure 2 Comparison of melting temperatures (Tm) of wild-type PfAgo and mutants;

[0059] Figure 3 This is a comparison of the 65°C cleavage activity of wild-type PfAgo and mutants. DETAILED DESCRIPTION

[0060] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0061] Example 1 Construction and synthesis of wild-type PfAgo plasmid vector and selection of mutants

[0062] The wild-type nucleotide sequence of PfAgo was searched in the NCBI database, and sent to Sangon Biotech for synthesis. After codon optimization, the pET28a(+) vector was selected, and the NdeI and XhoI double restriction sites were selected to construct the pET28a-PfAgo plasmid. The plasmid product synthesized by the company was transformed into the competent state of Escherichia coli Ecoli-BL21 (DE3), coated on a plate (containing 50 μg / ml kanamycin), and inverted overnight culture at 37°C. A single colony was picked up the next day for sequencing to verify whether the plasmid sequence was correct. After the alignment was correct, the plasmid was extracted. The plasmid extraction steps refer to the instructions of the Axygen plasmid extraction kit. The plasmid concentration was determined by Nano-300.

[0063] The wild-type (WT) amino acid sequence of the Argonaute protein (PfAgo) from the thermophilic prokaryotic bacterium kurthia massiliensis involved in the present invention is shown in SEQ ID NO.4.

[0064] PfAgo was designed from scratch using the existing GNN model, supplemented by screening methods such as Alpahfold to select PfAgo proteins designed from scratch by AI.

[0065] Example 2 Expression of de novo designed proteins and determination of cleavage activity

[0066] Transform the pET28a-PfAgo plasmid into Escherichia coli BL21 (DE3), inoculate a single colony into LB liquid medium containing 50μg / mL kanamycin, and culture on a shaker at 37°C and 220rpm. When the bacterial OD600 reaches 0.6-0.8, add a final concentration of 0.5mM IPTG (isopropyl-β-D-thiogalactoside) and move to an 18°C ​​shaker overnight. Collect the bacteria by centrifugation at 4000rpm for 15-20min, wash the bacteria with Buffer A (20mM Tris-HCl pH 7.5, 500mM NaCl, 10mM imidazole), resuspend the bacteria in Buffer A, and ultrasonically disrupt them. Centrifuge at 13,300rpm for 45min and collect the supernatant. After filtering the supernatant, Ni-NTA purification is performed. Use 10mM, 20mM and 250mM imidazole to wash 1 column volume each, collect the eluted fractions containing high-purity target protein, and replace the buffer with ultrafiltration to Buffer B (20mM Tris-HCl pH 7.5, 500mM NaCl). Collect the purified protein and identify the purity using SDS-polyacrylamide gel ( Figure 1 ). The protein was divided into small portions, quick-frozen in liquid nitrogen, and stored at -80°C.

[0067] Design a 16nt gDNA sequence and a 30nt target DNA sequence and send them to the company for synthesis. The sequences of gDNA and target DNA are shown in Table 1.

[0068] Table 1. Sequences of gDNA and target DNA

[0069]

[0070] Prepare reaction buffer (containing 15mM Tris-HCl pH 8.8, 250mM NaCl), add MnCl2 with a final concentration of 20μM, 100nM PfAgo, 1μM synthetic gDNA and 1μM complementary single-stranded DNA target nucleic acid to the reaction buffer, react at 65°C for 120min at an excitation wavelength of 490nm and an emission wavelength of 550nm, measure the fluorescence value at the end point, and perform three replicates for each experiment.

[0071] The data of enzyme activity improvement are shown in Table 2. After screening, three de novo designed proteins with improved enzyme activity were obtained: Pf-19, Pf-65 and Pf-71.

[0072] Table 2. Enzyme activity improvement

[0073]

[0074] Figure 3A bar chart showing the improvement of protein activity by de novo design of PfAgo. The results show that the activity of mutant proteins can be increased by up to 7.0 times compared with wild-type proteins. It can also be used for gene detection in vitro, showing good application prospects.

[0075] Example 3 Tm test of wild-type PfAgo and de novo designed proteins

[0076] 1.0 μL SSYPROOrange Dye (SUPELCO) was added to 49 μL lysis buffer (25 mM Tris-HCl, 500 mM NaCl, pH 7.4). Then 1 μL of the diluted dye was mixed with 19 μL of 0.1 mg / mL protein. Tm testing was then performed using a LightCycler 480 Instrument II (Roche). The reaction mixture was first brought to 25°C and then heated at 0.05°C s -1 The temperature was raised to 99°C at a speed of 100 °C and maintained for 2 min. Data were processed using Protein Thermal Shift software. Figure 2 are the Tm values ​​of the wild type and designed proteins.

[0077] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A de novo designed high temperature endonuclease protein PfAgo or its active fragment, variant form, derivative protein, characterized in that: The high temperature endonuclease protein PfAgo has an amino acid sequence shown in any one of SEQ ID NOs. 1 to 3, or an amino acid sequence that has at least 50% identity with SEQ ID NOs. 1 to 3.

2. An isolated polynucleotide, characterized in that It encodes the de novo designed protein PfAgo or its active fragment, variant form, or derivative protein as described in claim 1.

3. A carrier, characterized in that It contains the isolated polynucleotide according to claim 2.

4. A host cell, characterized in that It contains the vector according to claim 3, or its nucleic acid contains the isolated polynucleotide according to claim 2.

5. A method for preparing a de novo designed high temperature endonuclease protein PfAgo or its active fragment, variant form, or derivative protein as claimed in claim 1, characterized in that: Cultivate the host cell according to claim 4 to express the mutant protein or its active fragment, variant form, or derivative protein.

6. Use of the de novo designed high-temperature endonuclease protein PfAgo or its active fragments, variant forms, and derivative proteins described in claim 1 in nucleic acid detection and gene editing.

7. An in vitro DNA cleavage system based on the de novo designed protein PfAgo or its active fragment, variant form, or derivative protein according to claim 1, characterized in that: include: (A) a guide DNA, wherein the guide DNA binds to a predetermined target site; and (B) a programmable nuclease Argonaute, wherein the programmable nuclease is the aforementioned PfAgo mutant protein or an active fragment, variant form, or derivative protein thereof; and (C) a target DNA having a nucleotide sequence that is largely complementary to the guide DNA.

8. A method for in vitro DNA cleavage based on the system of claim 7, characterized in that: The specific steps are as follows: S1. Formation of PfAgo-guide complex based on PfAgo and gDNA; S2. The PfAgo-guide complex is brought into contact with the target DNA, and the PfAgo-guide complex cuts the target DNA at a specific site.

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