A de novo designed mesophilic endonuclease protein and preparation method and application thereof

Abstract

The application discloses a de novo designed mesophilic endonuclease protein and a preparation method and application thereof, and relates to a mutant protein with significantly improved activity of KmAgo enzyme, which is a de novo designed multi-point mutant protein or an active fragment, a variant form or a derivative protein of the mesophilic nuclease Argonaute based on kurthia massiliensis. The mesophilic endonuclease protein KmAgo has nuclease activity in a temperature range of 10-80 DEG C. The catalytic activity of the mesophilic endonuclease protein KmAgo is significantly improved compared with that of the wild type, and the mesophilic endonuclease protein KmAgo has better high-temperature thermal stability.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering technology, and relates to a de novo-designed mesophilic endonuclease protein, its preparation method, and its application. Background Technology

[0002] Argonaute (Ago) proteins, as important components of the RNA-induced silencing complex (RISC), play a crucial role in the cleavage and recruitment of small RNA molecules, and are a protein family that has attracted considerable attention since the discovery of RNA interference (RNAi). Currently, Argonaute proteins are found to be widely distributed in eukaryotes and prokaryotes, with different functions in each species. In eukaryotes, Ago proteins mainly participate in the RNAi process, playing a vital role in the formation of the RISC complex; while in prokaryotes, Ago proteins primarily function as host defense mechanisms, participating in bacterial defense mechanisms against foreign gene invasion and in host replication and damage repair.

[0003] Prokaryotic-derived Argonaute proteins (pAgos) are more diverse in function and structure than eAgos, but their physiological functions have long been elusive. Early research mainly focused on pAgos from thermophilic organisms. Except for MpAgo, which prefers to use 5'-hydroxylated (5'OH) RNA-guided gDNA to cleave target single-stranded DNA (ssDNA) and target RNA, other thermophilic pAgos prefer to use 5'-phosphorylated (5'P) gDNA to cleave target ssDNA and / or target RNA. Thermophilic pAgos exhibit only low levels of gDNA-guided target ssDNA and / or target RNA cleavage activity under mesophilic conditions, limiting the application development of thermophilic pAgos. Recent research has begun to focus on mesophilic pAgos, aiming to find pAgos that can effectively cleave target DNA and / or target RNA under mesophilic conditions. Similar to the commonly used CRISPR-Cas9 and CRISPR-Cas12a / 13a, some have suggested using pAgos as a next-generation genome editing tool. However, the KmAgo protein exhibits low endonuclease activity under mesophilic conditions, which significantly limits its application in gene editing and nucleic acid detection.

[0004] Traditional protein engineering techniques, such as directed evolution which involves small perturbations to natural protein sequences, are essentially trial-and-error methods. Without high-throughput screening, they are inefficient and struggle to create proteins with novel structures and functions. However, with the rapid development of computer technology and AI, the application of computers in enzyme engineering has continuously expanded the scope of enzyme sequence spatial exploration. Methods that determine amino acid sequences computationally based on structural and functional requirements can be used for de novo protein design and are increasingly applied to the modification of existing proteins, representing a crucial area for advancement.

[0005] Therefore, there is an urgent need to develop a method to obtain a KmAgo mutant with high catalytic activity for this protein. Furthermore, modifying the catalytic activity of KmAgo will further advance the development of de novo protein design technology. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a de novo-designed mesophilic endonuclease protein, its preparation method and application, which significantly improves enzyme catalytic activity and has very good thermal stability.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] 1. A de novo-designed mesophilic endonuclease protein KmAgo or its active fragment, variant form, or derived protein, wherein the mesophilic endonuclease protein KmAgo is selected from the amino acid sequences shown in SEQ ID NO. 1 to 11 (specifically AP1, AP2, AP7, AP10, AP14, AP16, AP21, AP22, AP23, AP24, and AP25), or an amino acid sequence having at least 50% identity with SEQ ID NO. 1 to 11; the mesophilic endonuclease is derived from the mesophilic prokaryote kurthiamassiliensis.

[0009] As one of the preferred technical solutions, the mesophilic endonuclease protein KmAgo is selected from the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.11, namely AP2, AP7, AP16, AP21, AP22, AP23, and AP25.

[0010] As a further preferred technical solution, the mesophilic endonuclease protein KmAgo is selected from the amino acid sequences shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.11, namely AP21, AP22, AP23, and AP25.

[0011] As a further preferred technical solution, the mesothermal endonuclease protein KmAgo is selected from the amino acid sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9, namely AP22 and AP23.

[0012] As one of the preferred technical solutions, the intermediate temperature is 10-80℃, more preferably 32-44℃, and even more preferably 37℃.

[0013] As one of the preferred technical solutions, the ratio of the enzyme activity Q1 of the mesophilic endonuclease protein KmAgo to the enzyme activity Q0 of the wild type is: Q1 / Q0 ≥4, preferably ≥5, more preferably ≥7, and most preferably ≥8.

[0014] As a further preferred technical solution, the ratio of the enzyme activity Q1 of the mesophilic endonuclease protein KmAgo to the enzyme activity Q0 of the wild type is 4.0 to 5.0, and more preferably 7.0 to 8.6.

[0015] As one of the preferred technical solutions, the active fragment, variant form, and derived protein include:

[0016] The enzyme retains directional single-strand DNA cleavage activity (cA1) after having one or more (e.g., typically 1-30, preferably 1-10, more preferably 1-6, even more preferably 1-3, best of all 1) amino acid residues deleted, inserted, and / or substituted on the sequences shown in SEQ ID NO. 1-11. This activity is significantly higher than the corresponding activity (cA0) of the wild-type KmAgo enzyme shown in SEQ ID NO. 12. The significantly higher activity is any value in the range of (cA1-cA0) / cA0 ≥ 10%-800%, for example ≥ 15%, ≥ 20%, ≥ 40%, ≥ 50%, ≥ 100%, ≥ 200%, or ≥ 600% or higher.

[0017] Protein modifications (which typically do not alter the primary structure) include: chemically derived forms of proteins in vivo or in vitro, such as acetylation or carboxylation; glycosylation; sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine); and proteins modified to improve their resistance to proteolysis or optimize their solubility.

[0018] The aforementioned protein or its active fragment forms a fusion protein or conjugate with other proteins or markers;

[0019] It contains one or more other mutations, thereby further enhancing the enzymatic activity of the de novo-designed mesophilic endonuclease protein KmAgo.

[0020] 2. An isolated polynucleotide encoding the aforementioned mesophilic endonuclease protein KmAgo or its active fragment, variant form, or derivative protein.

[0021] 3. A carrier containing the aforementioned isolated polynucleotides.

[0022] 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 it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements.

[0023] 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 identical to or a degenerate variant of the nucleotide sequence encoding the mesophilic endonuclease protein KmAgo in the embodiments of the present invention.

[0024] 4. A host cell containing the aforementioned vector, or whose nucleic acid contains the aforementioned isolated polynucleotide.

[0025] As one of the preferred technical solutions, the host cell includes cells derived from the following microorganisms:

[0026] Saccharomyces cerevisiae, Pichia pastoris, Saccharo mycesmonacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipites, Candida shehatae, Candida tropicalis, and Escherichia coli.

[0027] The de novo designed protein of the present invention can be obtained by conventional recombinant transformation methods in the art, and the de novo designed protein can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeabilization, sonication, high-pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. The host cell-expressible mesophilic endonuclease protein KmAgo of the present invention can be obtained.

[0028] As a further preferred technical solution, host cells are cultured under suitable expression conditions to express the mesophilic endonuclease protein KmAgo; and

[0029] The mesophilic endonuclease protein KmAgo was isolated.

[0030] The obtained mesophilic endonuclease protein KmAgo can be optionally purified to obtain a purer protein product.

[0031] As a further preferred technical solution, the suitable expression conditions include conventional techniques in the art, and the purification techniques include nickel column purification, ion exchange chromatography, etc.

[0032] 5. The aforementioned method for preparing a de novo-designed mesophilic endonuclease protein KmAgo or its active fragment, variant form, or derived protein, and culturing the aforementioned host cells to express the aforementioned mesophilic endonuclease protein KmAgo or its active fragment, variant form, or derived protein.

[0033] 6. The application of the aforementioned de novo-designed mesophilic endonuclease protein KmAgo or its active fragment, variant forms, and derived proteins in nucleic acid detection and gene editing.

[0034] 7. A system for in vitro DNA cleavage based on the aforementioned de novo-designed mesophilic endonuclease protein KmAgo or its active fragment, variant forms, or derived proteins, comprising:

[0035] (A) Guide DNA (gDNA), said guide DNA (gDNA) targeting and binding to a predetermined target site; and

[0036] (B) A programmable endonuclease Argonaute (KmAgo), wherein the programmable endonuclease is the aforementioned KmAgo mutant protein or its active fragment, variant form, or derived protein; and

[0037] (C) Target DNA, which has a nucleotide sequence that is largely complementary to the guide DNA.

[0038] As one of the preferred technical solutions, the length of the guide DNA is 21-45 nt, more preferably 15-21 nt, and even more preferably 16-18 nt.

[0039] As one of the preferred technical solutions, the guide DNA and the target nucleic acid have a reverse complementary fragment.

[0040] 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.

[0041] As a further preferred technical solution, the fluorescent group includes: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or combinations thereof, with FAM being even more preferred.

[0042] As a further preferred technical solution, the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof, with BHQ being even more preferred.

[0043] 8. A method for in vitro DNA cleavage based on the aforementioned system, the specific steps of which are as follows:

[0044] S1. Formation of a KmAgo-guided complex based on KmAgo and gDNA;

[0045] S2. Contact the KmAgo-guided complex with the target DNA, wherein the KmAgo-guided complex cleaves the target DNA at a specific site.

[0046] As one of the preferred technical solutions, the nuclease activity of the KmAgo and KmAgo-guided complex requires the presence of at least one component 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.

[0047] As a further preferred technical solution, the cation of the KmAgo and KmAgo-guided complex is Mn. 2 + .

[0048] As one of the further preferred technical solutions, the concentration of divalent metal ions is 10 μM to 3 mM, more preferably 10 μM to 200 mM, and even more preferably 20 μM.

[0049] The beneficial effects of this invention are as follows:

[0050] Through extensive and in-depth research and screening, this invention has obtained mutant proteins that significantly improve the activity of KmAgo enzymes. These mutant proteins are de novo designed multi-point mutant proteins or their active fragments, variant forms, and derived proteins based on the mesophilic nuclease Argonaute (KmAgo, wild-type sequence shown in SEQ ID NO.12) of Kurthia massiliensis.

[0051] This invention utilizes a GNN model to learn the structural information of different pAgo proteins, and employs screening methods such as Alpahfold to select AI-designed de novo Ago proteins. Through screening, mutants with enhanced enzyme catalytic activity are obtained. In this invention, 11 highly active mutants were ultimately selected, chosen from the amino acid sequences shown in SEQ ID NO. 1–11 (specifically AP1, AP2, AP7, AP10, AP14, AP16, AP21, AP22, AP23, AP24, and AP25), or amino acid sequences with at least 50% identity to SEQ ID NO. 1–11.

[0052] The mesophilic endonuclease protein KmAgo described in this invention exhibits nuclease activity within a temperature range of 10 to 80°C, preferably 32 to 44°C, and even more preferably at 37°C. The mesophilic endonuclease protein KmAgo exhibits significantly enhanced catalytic activity compared to the wild type and demonstrates better high-temperature thermal stability.

[0053] The mesophilic endonuclease protein KmAgo significantly enhances the activity of targeted single-stranded DNA cleavage, with an activity increase of 400% to 860%, thus aiding in pathogen detection, genotyping, and disease progression monitoring. The method of this invention can perform site-specific modification of extracellular genetic material, and therefore can be effectively applied in biotechnology fields such as nucleic acid detection and gene editing. This enhances the application potential of this protein in the field of nucleic acid detection technology and also lays the foundation for the development of new gene manipulation tools.

[0054] The main advantages of this invention are as follows:

[0055] 1. The mutant strain obtained by screening in this invention exhibits significantly improved enzyme catalytic activity compared to the wild type, up to a maximum of 8.6 times;

[0056] 2. The thermal stability of the mutant of the present invention is not adversely affected by the mutation and maintains very good thermal stability;

[0057] 3. The mutants of this invention have potential applications in nucleic acid detection technology and in the development of gene editing tools. Attached Figure Description

[0058] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following drawings are provided for illustration.

[0059] Figure 1 SDS-PAGE electrophoresis images of wild-type KmAgo and mutants, where M: Marker; Lane 1: purified WT sample; Lanes 2-12: purified mutant protein samples;

[0060] Figure 2 Substrate shearing efficiency versus time for wild-type KmAgo and mutant;

[0061] Figure 3 This is a comparison of the cleavage activity between wild-type KmAgo and the mutant. Detailed Implementation

[0062] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0063] Example 1: Construction and synthesis of wild-type KmAgo plasmid vector and selection of mutants

[0064] Nucleotide sequences for the wild-type KmAgo strain were searched in the NCBI database and synthesized by Sangon Biotech. After codon optimization, the pET28a(+) vector was selected, and NdeI and XhoI double restriction enzyme sites were chosen. The synthesized plasmid product was transformed into E. coli Ecoli-BL21(DE3) competent cells, plated on plates (containing 50 μg / ml kanamycin), and incubated overnight at 37°C inverted position. The next day, single colonies were picked and sent for sequencing to verify the plasmid sequence. After confirmation of correct alignment, the plasmid was extracted. The plasmid extraction procedure followed the instructions of the Axygen plasmid mini-prep kit. The plasmid concentration was determined using a Nano-300 analyzer.

[0065] The wild-type (WT) amino acid sequence of the Argonaute protein (KmAgo) from the mesophilic prokaryotic bacterium *Kurthia massiliensis* involved in this invention is shown in SEQ ID NO.12.

[0066] KmAgo was designed de novo using existing GNN models, and AI-designed de novo KmAgo proteins were selected using screening methods such as Alpahfold.

[0067] Example 2: Expression and cleavage activity assay of de novo designed proteins

[0068] pET28a-KmAgo plasmid was transformed into *E. coli* BL21(DE3). Single colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured in shake flasks at 37°C and 220 rpm. When the OD600 of the cells reached 0.6-0.8, 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration, and the culture was transferred to a shake flask at 18°C ​​and incubated overnight. The cells were collected by centrifugation at 4000 rpm for 15-20 min, washed with Buffer A (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM imidazole), and then resuspended in Buffer A and sonicated. The cells were centrifuged at 13,300 rpm for 45 min, and the supernatant was collected. After filtration, the supernatant was purified using Ni-NTA. Wash one column volume each with 10 mM, 20 mM, and 250 mM imidazole, collect the elution fraction containing high-purity target protein, and ultrafilter to Buffer B (20 mM Tris-HCl pH 7.5, 500 mM NaCl). Collect the purified protein and determine its purity using an SDS-polyacrylamide gel electrophoresis gel. Figure 1 The protein was divided into small portions, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0069] A 16nt gDNA sequence and a 30nt target DNA sequence were designed and sent to the company for synthesis. The sequences of the gDNA and target DNA are shown in Table 1.

[0070] Table 1. Sequences of gDNA and target DNA

[0071]

[0072] Prepare a reaction buffer (containing 15 mM Tris-HCl pH 8.8 and 250 mM NaCl). Add a final concentration of 20 μM MnCl2, 100 nM KmAgo, 1 μM synthesized gDNA, and 1 μM complementary single-stranded DNA target nucleic acid to the reaction buffer. React at 37°C for 120 min at an excitation wavelength of 490 nm and an emission wavelength of 550 nm. Measure the fluorescence value at the endpoint. Perform three replicates for each experiment. Figure 3 As shown.

[0073] Prepare a reaction buffer (containing 15 mM Tris-HCl pH 8.8 and 250 mM NaCl). Add a final concentration of 20 μM MnCl2, 100 nM KmAgo, 1 μM synthesized gDNA, and 1 μM complementary single-stranded DNA target nucleic acid to the reaction buffer. Incubate at 37°C for 30 min. Take 6-10 μL of sample and add loading buffer (containing 95% (deionized) formamide, 0.5 mmol / L EDTA, 0.025% bromophenol blue, and 0.025% xylene blue) at a 1:1 volume ratio. Perform electrophoresis on a 16% nucleic acid Urea-PAGE gel. Figure 2 As shown.

[0074] result

[0075] The enzyme activity enhancement data are shown in Table 2. After screening, 11 de novo designed proteins with enhanced enzyme activity were obtained: AP1, AP2, AP7, AP10, AP14, AP16, AP21, AP22, AP23, AP24 and AP25.

[0076] Table 2. Enzyme activity enhancement

[0077]

[0078] Figure 3 A bar chart showing the increased activity of de novo KmAgo protein design was generated. The results indicate that the mutant protein activity can be increased by up to 8.6 times compared to the wild-type protein. Furthermore, it can be used for in vitro gene detection, demonstrating promising application prospects.

[0079] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended 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 to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A de novo-designed mesophilic endonuclease protein Km Ago, characterized by... The mesothermal endonuclease protein Km Ago is the amino acid sequence shown in SEQ ID NO. 9; the mesophilic endonuclease is derived from mesophilic prokaryotes. kurthia massiliensis .

2. An isolated polynucleotide, characterized in that, It encodes the mesophilic endonuclease protein of claim 1. Km Ago.

3. A carrier, characterized in that, It contains the isolated polynucleotide as described in claim 2.

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

5. The de novo designed mesophilic endonuclease protein according to claim 1 Km The method for preparing Ago is characterized by, Culture the host cells of claim 4 to express the mesophilic endonuclease protein of claim 1. Km Ago.

6. The de novo designed mesophilic endonuclease protein according to claim 1 Km Applications of Ago in the preparation of nucleic acid detection and gene editing reagents.

7. A mesophilic endonuclease protein designed de novo according to claim 1 Km Ago's in vitro DNA cleavage reagent is characterized by... include: (A) Guide DNA, which targets and binds to a predetermined target site; and (B) Argonaute, a programmable endonuclease, wherein the programmable endonuclease is as described in claim 1. Km Ago; and (C) Target DNA, which has a nucleotide sequence that is largely complementary to the guide DNA.

8. A method for in vitro DNA cutting based on the reagent of claim 7, characterized in that, The specific steps are as follows: S1. Based on Km Ago and gDNA formation Km Ago Guide Complex; S2. Order Km The Ago guide complex contacts the target DNA, Km The Ago guide complex cuts target DNA at specific sites.

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