Escherichia coli toxic protein, use thereof and prokaryotic expression method

Abstract

The present invention provides a method for efficiently expressing and purifying a cytotoxic protein HNH having endonuclease activity, and demonstrates the endonuclease activity of HNH. The implementation of the present invention not only optimizes the expression and purification method of HNH, but also provides a potential tool enzyme.

Description

[0001] Technical Field

[0002] This invention relates to the field of enzyme and protein expression and purification methods, and particularly to a method for expressing and purifying the toxic protein HNH, which has endonuclease activity. Background Technology

[0003] HNH enzymes, also known as "ββα-me" endonucleases, are a family of enzymes that share a highly similar catalytic motif—the ββα topology. This structure consists of 30 to 40 amino acids and is responsible for the binding and cleavage of nucleic acids. The structure is characterized by three conserved amino acid His-Asn-His residues (HNH) folding into a β-sheet with two antiparallel β-sheets, an α-helix, and a β-α-metal topology that binds a divalent metal cation, ultimately folding into the ββα-metal topology.

[0004] The sequence conservation of HNH proteins is reflected in the conserved His residue (corresponding to the first letter "H" in HNH) at the end of the β1 chain, the binding of metal ions to the highly conserved Asn / His residues within the α-helix (corresponding to the last letter "H" in HNH), and the conserved Asn residue (corresponding to the letter "N" in HNH) at the beginning of the β2 chain. The highly conserved Asn typically forms hydrogen bonds with the main chain atoms adjacent to the catalytic His residue, thus playing a crucial structural role in fixing the position and orientation of general bases. When HNH nucleases function as monomers, they create single nicks in nucleic acids to degrade exogenous or host genomes (Nickase endonucleases). When they function as homodimers, they introduce double-strand DNA breaks for DNA restriction, integration, recombination, and repair.

[0005] Previous studies have shown that when expressing HNH proteins in prokaryotes, *E. coli* can be cultured, but after induction, the bacterial culture gradually becomes clear, suggesting that HNH proteins are cytotoxic. Therefore, establishing a non-cytotoxic expression and purification system is crucial to improving the expression efficiency of HNH proteins. Furthermore, the HNH protein family has many different sequences; identifying new HNH proteins with endonuclease activity can further enrich the number of enzyme tools, providing more options for enzyme modification and optimization. Summary of the Invention

[0006] To address the problems existing in the prior art, embodiments of the present invention provide a method for co-expressing HNH protein with ATPase protein within the same open reading frame, thereby obtaining soluble target HNH protein.

[0007] The primary objective of this invention is to provide an HNH toxic protein expression vector, wherein the vector contains an ATPase coding frame and an HNH coding frame, and the ATPase coding frame and the HNH coding frame can be expressed simultaneously and independently.

[0008] Furthermore, the HNH toxic protein expression vector is constructed by inserting the ATPase coding frame and the HNH coding frame into any prokaryotic expression vector.

[0009] Furthermore, the HNH toxic protein expression vector is composed of the positions of the ATPase coding frame and the HNH coding frame inserted into any prokaryotic expression vector.

[0010] Furthermore, the ATPase coding frame and the HNH coding frame are both derived from the Escherichia coli Retron-Eco4 system.

[0011] Furthermore, the ATPase coding frame and the HNH coding frame are both derived from the same bacterial Retron-Eco4 system.

[0012] Furthermore, the ATPase coding frame and the HNH coding frame are both derived from the same bacterial Retron-Eco4 system.

[0013] Furthermore, the protein encoded by the ATPase coding frame and the protein encoded by the HNH coding frame interact, and the protein encoded by the ATPase coding frame can inhibit the cytotoxicity of the protein encoded by the HNH coding frame.

[0014] Furthermore, the interaction between the protein encoded by the ATPase coding frame and the protein encoded by the HNH coding frame is unstable or the binding force is weak, and the two will separate during molecular sieve chromatography.

[0015] In a preferred embodiment, an ATPase coding frame or an HNH coding frame is connected to a tag protein coding frame, the tag protein coding frame and the ATPase coding frame or HNH coding frame together express a fusion protein containing a tag protein and ATPase or HNH, the tag protein being located at the 5' or 3' end of the ATPase coding frame or HNH coding frame.

[0016] In a preferred embodiment, the tag protein may be one of 6×His, FLAG, or HA.

[0017] In a preferred embodiment, the HNH coding frame is located downstream of the ATPase coding frame, with a spacer sequence between them, which allows the HNH coding frame and the ATPase coding frame to be expressed independently.

[0018] In a preferred embodiment, the HNH coding frame, ATPase coding frame, spacer sequence, and tag protein coding frame are formed as follows: F-ATPase-B-HNH or ATPase-B-HNH-F, where F is the coding frame of the tag protein, B is the spacer sequence, preferably F is 6×His, and B is RBS (ribosome binding site).

[0019] In a preferred embodiment, the ATPase coding frame encodes the amino acid sequence shown in SEQ ID NO2, and the HNH coding frame expresses the amino acid sequence shown in SEQ ID NO1.

[0020] In a preferred embodiment, the structure of the HNH toxic protein expression vector containing the HNH coding frame, ATPase coding frame, spacer sequence, and tag protein coding frame is as shown in sequence SEQ ID NO.3 or SEQ ID NO.4, preferably SEQ ID NO.3.

[0021] In a preferred embodiment, the above sequence is inserted between the BamHI / HindIII restriction sites following the first T7 promoter of the pCDFDuet vector.

[0022] A method for expressing and purifying HNH toxic protein, the method comprising the following steps:

[0023] 1) Simultaneous and independent expression of the HNH coding frame and the ATPase coding frame in the same cell.

[0024] 2) Preliminary purification of the expression product.

[0025] 3) The initially purified product is further purified.

[0026] In a preferred embodiment, in step 1), the HNH coding frame and the ATPase coding frame are expressed independently and simultaneously in the same vector.

[0027] Furthermore, the ATPase coding frame and HNH coding frame mentioned in step 1) are both ATPase coding frames and HNH coding frames from the Escherichia coli Retron-Eco4 system.

[0028] Furthermore, the ATPase coding frame and HNH coding frame mentioned in step 1) are both from the same bacterial Retron-Eco4 system.

[0029] Furthermore, the ATPase coding frame and HNH coding frame mentioned in step 1) are both from the same bacterial Retron-Eco4 system.

[0030] Furthermore, the protein encoded by the ATPase coding frame and the protein encoded by the HNH coding frame described in step 1) interact, and the protein encoded by the ATPase coding frame can inhibit the cytotoxicity of the protein encoded by the HNH coding frame.

[0031] Furthermore, the interaction between the protein encoded by the ATPase coding frame and the protein encoded by the HNH coding frame described in step 1) is unstable or the binding force is weak, and the two will separate during molecular sieve chromatography.

[0032] Further, in step 1), an ATPase coding frame or an HNH coding frame is connected to a tag protein coding frame, and the tag protein coding frame together with the ATPase coding frame or the HNH coding frame expresses a fusion protein containing a tag protein and ATPase or HNH, wherein the tag protein is located at the 5' or 3' end of the ATPase coding frame.

[0033] In a preferred embodiment, the tag protein may be one of 6×His, FLAG, or HA.

[0034] In a preferred embodiment, the HNH coding frame is located downstream of the ATPase coding frame, with a spacer sequence between them, which allows the HNH coding frame and the ATPase coding frame to be expressed independently.

[0035] In a preferred embodiment, the HNH coding frame, ATPase coding frame, spacer sequence, and tag protein coding frame are formed as follows: F-ATPase-B-HNH or ATPase-B-HNH-F, where F is the coding frame of the tag protein, B is the spacer sequence, preferably F is 6×His, and B is RBS (ribosome binding site).

[0036] In a preferred embodiment, the ATPase coding frame encodes the amino acid sequence shown in SEQ ID NO2, and the HNH coding frame expresses the amino acid sequence shown in SEQ ID NO1.

[0037] In a preferred embodiment, the structure of the HNH toxic protein expression vector containing the HNH coding frame, ATPase coding frame, spacer sequence, and tag protein coding frame is as shown in sequence SEQ ID NO.3 or SEQ ID NO.4, preferably SEQ ID NO.3.

[0038] In a preferred embodiment, the above sequence is inserted between the BamHI / HindIII restriction sites following the first T7 promoter of the pCDFDuet vector.

[0039] In a preferred embodiment, step 3) involves purification using gel filtration chromatography.

[0040] An endonuclease having an amino acid sequence as shown in sequence SEQ ID NO.1.

[0041] In a preferred embodiment, the sequence of the endonuclease is the amino acid sequence shown in SEQ ID NO.1.

[0042] Furthermore, the endonuclease possesses nickase activity for cutting single-stranded DNA.

[0043] The present invention has the following beneficial effects:

[0044] 1. A novel vector and purification method for expressing and purifying toxic proteins are provided, which improves the expression efficiency of toxic proteins.

[0045] 2. A novel enzyme with nickase activity for cutting single-stranded DNA is provided. Attached Figure Description

[0046] The method of the present invention and its beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] Figure 1 : Construct expression plasmids required for cytotoxicity experiments.

[0048] Figure 2 Results of cytotoxicity experiments. The bacterial concentration values ​​gradually decrease from top to bottom, and the image shown represents three replicate experiments. (A) HNH exhibits cytotoxicity; (B) ATPase inhibits HNH cytotoxicity.

[0049] Figure 3 Results of Co-IP experiments of ATPase and HNH. (A) SDS-PAGE detection results of co-expression of ATPase-FLAG and HNH-HA; (B) Western blot results of ATPase-FLAG and HNH-HA.

[0050] Figure 4 Construction of ATPase and HNH co-expression plasmid.

[0051] Figure 5Results of co-expression of ATPase and HNH. (A) SDS-PAGE results of co-expression of ATPase and HNH with the tag band on HNH; (B) SDS-PAGE results of co-expression of ATPase and HNH with the tag band on ATPase; (C) Superdex 200 gel filtration chromatography purification results of the co-expressed ATPase and HNH product.

[0052] Figure 6 Results of HNH endonuclease activity. (A) Nickase activity exists in 100 nM HNH protein; (B) Effect of different divalent cations on HNH enzyme activity; (C) Effect of different nucleotides on HNH enzyme activity; (D) Schematic diagram of endonuclease experiment.

[0053] Figure 7 Changes in bacterial culture after IPTG induction: The bacteria in the left centrifuge tube became clear after 1 hour of induction with 0.2 mM IPTG, while the uninduced bacteria on the right remained turbid after 1 hour. Detailed Implementation

[0054] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing specific embodiments only and is not intended to limit the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials in the following embodiments are all purchased from conventional biochemical reagent stores.

[0055] The amino acid sequences of the HNH protein and ATPase protein described in the examples correspond to SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0056] SEQ ID NO.1(HNH):

[0057] MILKRINKTAEDQFLINFKAQNPNGTWDEFRNHEQGILYKRLKQHICNDQMYLCAYCEIDLDRENEHEIKVEHFKSKSG

[0058] SLPGGSNWHLEWSNLLAVCLGGTNTGDDFELPANLSCDSYKSHYEDKNKINDKDWTGKILLPLTLPDAHNFFTFEKVTG

[0059] KLLPNESYCNTISIDGKPAAETLSIVTKTIEVLNLNCSRLNNARRKLLFHFNNCARERNLRKLHNLLLQWNQGEPKFFQ

[0060] TTRDIIIRDDRICQGLLNGTIRYYPYDVPDYA

[0061] SEQ ID NO.2(ATPase):

[0062] MEQNLPSRITKLIKKSESGDFASSYQLYKVFGSKEYGVEPDEKMSDYFKELSAKQLEGGQLRVADIHLENYKGFESLIM

[0063] DFSMKKNSTILVGNNGCGKSTILDAIQKGLTHLSSRLSTRSHNGDGIEKHELRKGQNYASIAINYDYMGIRFPMIIATT

[0064] EPGYEDRAKSNYSGINELGSIFKTAHSINPNVSFPLIAMYTVERANDVSTRDIENSEEIKEAQIWDKFKAYNKSLTGKA

[0065] DFKLFFRWFKELIEIENSDNADITALRAEIRAKEKDLDNPLLKALLAENKNSETTKKLLEDHQNSLKVLKEKLNSYYSV

[0066] NSKTLHTVEDAMYSFLPGFSNLKLQRAPLDLIVDKNNVSLSVLQLSQGEKTILALIADIARRLTLLNPNSVNPLDGTGI

[0067] VLIDEIDLHLHPSWQQNIIPRLEKTFKNIQFIVTTHSPQVCHTIDSQNIWLLKNGQKFKAPKGVRGAISSWVLENLFEV

[0068] AQRPPEDKYTKLLQEYKNLVFSEKYASEDARKLGATLSQHFGPDDETLVELKLEIEKRIWEDDFEKDYKDDDDKSEQ ID NO.3(6XHis-ATPase-RBS-HNH):

[0069] catcatcatcatcacagcggcgaaaacctgtattttcagggcgctagcGGATCCATGGAACAGAACTTACCGAGTA

[0070] GAATAACTAATTAATTAAAAAATCGGAAAGCGGCGATTTTGCTTCTTCGTATCAGCTTTATAAAGTATTTGGCTCGAA

[0071] AGAGTACGGAGTTGAGCCAGATGAAAAAATGTCTGATTACTTCAAAGAATTATCAGCTAAACAGTTAGAAGGTGGTCAA

[0072] CTGAGAGTTGCTGATATTCATTTGGAAAACTACAAAGGGTTTGAGTCCCTAATGGATTTTTCCATGAAAAAAACT

[0073] CTACAATTTTAGTAGGAAATAATGGCTGTGGGAAAatcgacGATTCTCGATGCAATCCAGAAGGGGTTAACACATCTATC

[0074] CTCGAGATTATCTACTCGCTCGCATAATGGCGATGGTATCGAAACATGAGTTAAAAAGGACAAAACTATGCATCG

[0075] ATCGCTATAAATTACGACTATATGGGAATACGTTTTCCTATGATCATAGCTACAACTGAACCTGGTTATGAGGATAGAG

[0076] CAAAAAGTAACTATAGCGGTATTAATGAGTTAGGAAGCATTTTCAAAACAGCCCATTCAATCCAAATGTTTCATT

[0077] TCCTTTAATTGCCAATGTATACAGTTGAAAGGGCTAATGACGTTTCTACTAGAGATATTGAAaattcAGAAGAAATTAA

[0078] GAAGCTCAAATCTGGGATAAATTCAAAGCATATAATAAGAGCCTGACAGGAAAGGCTGATTTCAAATTATTCTTCAGAT

[0079] GGTTTAAAGAACTAATAGAAATCGAAAAACTCTGATAACGCTGATATAACAGCATTAAAGAGCAGAGATTCGTGCTAAAA

[0080] AAAAGACTTAGACAATCCATTGCTAAAAGCTCTTCTGGCAGAGAATAAAAAATTCTGAGACTACTAAAAAAATTGTTAGAA

[0081] GATCATCAGAACTCTCTGAAAGTTTTAAAAGAGAAATTAAATAGCTATTATTCAGTCAATAGTAAAACATTACACACTG

[0082] TTGAAGATGCAATGTATTCTTTCCTTCCTGGTTTTAGCAACCTTAAACTTCCAAAGGGCGCCTCTTGATCTGATAGTGGA

[0083] TAAGAATAATGTTTCCTTAAGTGTTCTGCAATTATCTCAAGGTGAAAAACCATTTTAGCATTAATTGCAGATATTGCT

[0084] CGTAGATTGACATTGTTAAACCCTAATAGTGTTAACCCTTTGGACGGTACTGGAATTGTATTAATCGATGAAATAGACC

[0085] TCCATTTACATCCATCATGGCAGCAAAATATTATTCCTCGTCTTGAGAAAACGTTTAAGAATATTCAATTTATAGTCAC

[0086] GACTCATAGTCCACAAGTTTGTCATACTATTGATAGTCAGAATATATGGTTGTTAAAGAATGGCCAAAAGTTTAAAGCA

[0087] CCGAAAGGAGTTAGAGGAGCAATATCTTCTTGGGTACTGGAGAACTTGTTCGAAGTTGCTCAAAGGCCGCCAGAGGATA

[0088] AGTACACAAAACTCTTACAGGAATATAAAAATTTAGTATTTTCAGAAAAATATGCTAGTGAAGATGCAAGAAAGCTAGG

[0089] TGCTACTTTATCCCAACATTTCGGACCGGATGATGAAACCTTAGTTGAGTTAaagctAGAAAATTGAAAAAAGAATTTGG

[0090] GAGGATGATTTTGAAAAGGATCAAGATTACAAGGACGACGATGACAAGTAAgaattcaatcaataggagaaatcaatGG

[0091] ATCCATGATTTTGAAAAGGATCAATAAAACTGCTGAAGATCAATTCTTAATTAATTTTAAAGCTCAAAATCCAAATGGG

[0092] ACTTGGGATGAATTTAGGAATCATGAACAAGGTATTTTATATAAGAGGTTAAAGCAACATATTTGCAATGATCAAATGT

[0093] ACCTTTGTGCGTATTGTGAGATAGATTTAGATCGAGAAAATGAACATGAAATAAAAGTAGAGCATTTCAAATCTAAATC

[0094] TGGTTCGCTCCCTGGTGGAAGTAACTGGCATTTAGAGTGGTCTAATCTCTTAGCTGTATGCCTAGGAGGTACAAATACA

[0095] GGTGATGATTTCGAATTACCAGCTAATCTAAGTTGTGATTCATATAAGTCACATTGAAGACAAAAATAAAATCAATG

[0096] ATAAAGACTGGACAGGCAAAATCCTGTTACCTTTAACGCTTCCGGATGCACACAATTTTTTTACTTTCGAGAAAGTTAC

[0097] AGGTAAGTTGCTACCTAATGAATCATACTGTAATACTATTAGCATAGATGGTAAACCTGCTGCAGAAACACTAAGTATT

[0098] GTAACTAAAACAATAGAAGTTCTAAATTTAACTGCAGCAGGCTAAATAATGCCAGAAGAAAACTGCTGTTTCACTTTA

[0099] ATAATTGCGCACGTGAAAGAAACTTGAGAAAGCTCCATAATCTATTTTACAATGGAATCAAGGTGAGCCTAAATTTTT

[0100] CCAAACGACACGAGATATAATTCGTGATGATAGAATCTGCCAAGGGTTACTGAACGGAACGATAAGATATTACCCA

[0101] TACGATGTTCCAGATTACGCTTAA

[0102] SEQ ID NO.4(ATPase-HNH-6×His):

[0103] ATGGAACAGAACTTACCGAGTAGAATAACTAAATTAAAAAATCGGAAAGCGGCGATTTTGCTTCTTCGTATCAGC

[0104] TTTATAAAGTATTTGGCTCGAAAGAGTACGGAGTTGAGCCAGATGAAAAAATGTCTGATTACTTCAAAGAATTATCAGC

[0105] TAAACAGTTAGAAGGTGGTCAACTGAGAGTTGCTGATATTCATTTGGAAAACTACAAAGGGTTTGAGTCCCTAAATG

[0106] GATTTTTCCATGAAAAAAAACTCTACAATTTTAGTAGGAAATAATGGCTGTGGGAAAtcgacGATTCTCGATGCAATCC

[0107] AGAAGGGGTTAACACATCTATCCTCGAGATTATCTACTCGCTCGCATAATGGCGATGGTATCGAAAAACATGAGTTAAG

[0108] AAAAGGACAAAACTATGCATCGATCGCTATAAATTACGACTATATGGGAATACGTTTTCCTATGATCATAGCTACAACT

[0109] GAACCTGGTTATGAGGATAGAGCAAAAGTAACTATAGCGGTATTAATGAGTTAGGAAGCATTTTCAAACAGCCCATT

[0110] CAATCAATCCAAATGTTTCATTTCCTTTAATTGCAATGTATACAGTTGAAAGGGCTAATGACGTTTCTACTAGAGATAT

[0111] TGAAaattcAGAAGAAATTAAAGAAGCTCAAATCTGGGATAAATTCAAAGCATATAATAAAGAGCCTGACAGGAAAGGCT

[0112] GATTTCAAATTATTCTTCAGATGGTTTAAAGAACTAATAGAAATCGAAAACTCTGATAACGCTGATATAACAGCATTAA

[0113] GAGCAGAGATTCGTGCTAAAGAAGAAGACTTAGACAATCCATTGCTAAAAGCTCTTCTGGCAGAGAATAAAAATTCTGA

[0114] GACTACTAAAAAAATTGTTAGAAGATCATCAGAACTCTCTGAAAGTTTTAAAAGAGAAATTAAATAGCTATTATTCAGTC

[0115] AATAGTAAAACATTACACACTGTTGAAGATGCAATGTATTCTTTCCTTCCTGGTTTTAGCAACCTTAAACTTCAAAGGG

[0116] CGCCTCTTGATCTGATAGTGGATAAGAATAATGTTTCCTTAAGTGTTCTGCAATTATCTCAAGGTGAAAAAACCATTTT

[0117] AGCATTAATTGCAGATATTGCTCGTAGATTGACATTGTTAAACCCTAATAGTGTTAACCCTTTGGACGGTACTGGAATT

[0118] GTATTAATCGATGAAATAGACCTCCATTTACATCCATCATGGCAGCAAAATATTATTCCTCGTCTTGAGAAAACGTTTA

[0119] AGAATATTCAATTTATAGTCACGACTCATAGTCCACAAGTTTGTCATACTATTGATAGTCAGAATATATGGTTGTTAAA

[0120] GAATGGCCAAAAGTTTAAAGCACCGAAAGGAGTTAGAGGAGCAATATCTTCTTGGGTACTGGAGAACTTGTTCGAAGTT

[0121] GCTCAAAGGCCGCCAGAGGATAAGTACACAAAACTCTTACAGGAATATAAAAATTTAGTATTTTCAGAAAAATATGCTA

[0122] GTGAAGATGCAAGAAAGCTAGGTGCTACTTTATCCCAACATTTCGGACCGGATGATGAAACCTTAGTTGAGTTAaagct

[0123] AGAAATTGAAAAAAGAATTTGGGAGGATGATTTTGAAAAGGATCAAGATTACAAGGACGACGATGACAAGTAAgaattc

[0124] aatcaataggagaaatcaatGGATCCATGATTTTGAAAAGGATCAATAAAACTGCTGAAGATCAATTCTTAATTAATTT

[0125] TAAAGCTCAAAATCCAAATGGGACTTGGGATGAATTTAGGAATCATGAACAAGGTATTTTATATAAGAGGTTAAAGCAA

[0126] CATATTTGCAATGATCAAATGTACCTTTGTGCGTATTGTGAGATAGATTTAGATCGAGAAAATGAACATGAAATAAAAG

[0127] TAGAGCATTTCAAATCTAAATCTGGTTCGCTCCCTGGTGGAAGTAACTGGCATTTAGAGTGGTCTAATCTCTTAGCTGT

[0128] ATGCCTAGGAGGTACAAATACAGGTGATGATTTCGAATTACCAGCTAATCTAAGTTGTGATTCATATAAGTCACATTAT

[0129] GAAGACAAAAATAAAATCAATGATAAAGACTGGACAGGCAAAATCCTGTTACCTTTAACGCTTCCGGATGCACACAATT

[0130] TTTTTACTTTCGAGAAAGTTACAGGTAAGTTGCTACCTAATGAATCATACTGTAATACTATTAGCATAGATGGTAAACC

[0131] TGCTGCAGAAACACTAAGTATTGTAACTAAAACATAGAAGTTCTAAATTTAAACTGCAGCAGGCTAAATAATGCCAGA

[0132] AGAAAACTGCTGTTTCACTTTAATAATTGCGCACGTGAAAGAAACTTGAGAAAGCTCCATAATCTATTATTACAATGGA

[0133] ATCAAGGTGAGCCTAAATTTTTCCAAACGACACGAGATATAATAATTCGTGATGATAGAATCTGCCAAGGGTTACTGAA

[0134] CGGAACGATAAGATATCACCACCACCACCACCACTAA

[0135] SEQ ID NO.5(pCDFDuet vector):

[0136] GGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGAGATATACCATGGGCAGC

[0137] AGCAGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAG

[0138] AAAGTAATCGTATTGTACACGGCCGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATT

[0139] CCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGGCAGATCTCAATTGGATATCGGCCGGCCACGC

[0140] GATCGCTGACGTCGGTACCCTCGAGTCTGGTTCTACTAGCGCAGCTTAATTAACCTAGGCTGCTGCCACCGCTGAGCAA

[0141] TAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAACCTCAGGCATTTGAGAAGCACA

[0142] CGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGA

[0143] ACCGACGACCGGGTCATCGTGGCCGGATCTTGCGGCCCCTCGGCTTGAACGAATTGTTAGACATTATTTGCCGACTACC

[0144] TTGGTGATCTCGCCTTTCACGTAGTGGACAAATTCTTCCAACTGATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTC

[0145] CAAGATAAGCCTGTCTAGCTTCAAGTATGACGGGCTGATACTGGGCCGGCAGGCGCTCCATTGCCCAGTCGGCAGCGAC

[0146] ATCCTTCGGCGCGATTTTGCCGGTTACTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGCTCATCGCCA

[0147] GCCCAGTCGGGCGGCGAGTTCCATAGCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAA

[0148] AGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTTTGTCAGCAAGATAGCCAGATCAATGTC

[0149] GATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCATTGCGCTGCCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGA

[0150] TAACGCCACGGAATGATGTCGTCGTGCACAACAATGGTGACTTCTACAGCGCGGAGAATCTCGCTCTCTCCAGGGGAAG

[0151] CCGAAGTTTCCAAAAGGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGCAAATC

[0152] AATATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAATGTACGGCCAGCAACGTCGGTTCGAGATGG

[0153] CGCTCGATGACGCCAACTACCTCTGATAGTTGAGTCGATACTTCGGCGATCACCGCTTCCCTCATACTCTTCCTTTTTC

[0154] AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT

[0155] AGCTAGCTCACTCGGTCGCTACGCTCCGGGCGTGAGACTGCGGCGGGCGCTGCGGACACATACAAAGTTACCCACAGAT

[0156] TCCGTGGATAAGCAGGGGACTAACATGTGAGGCAAAACAGCAGGGCCGCGCCGGTGGCGTTTTTCCATAGGCTCCGCCC

[0157] TCCTGCCAGAGTTCACATAAACAGACGCTTTTCCGGTGCATCTGTGGGAGCCGTGAGGCTCAACCATGAATCTGACAGT

[0158] ACGGGCGAAACCCGACAGGACTTAAAGATCCCCACCGTTTCCGGCGGGTCGCTCCCTCTTGCGCTCTCCTGTTCCGACC

[0159] CTGCCGTTTACCGGATACCTGTTCCGCCTTTCTCCCTTACGGGAAGTGTGGCGCTTTCTCATAGCTCACACACTGGTAT

[0160] CTCGGCTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTAAGCAAGAACTCCCCGTTCAGCCCGACTGCTGCGCCTTAT

[0161] CCGGTAACTGTTCACTTGAGTCCAACCCGGAAAAGCACGGTAAAACGCCACTGGCAGCAGCCATTGGTAACTGGGAGTT

[0162] CGCAGAGGATTTGTTTAGCTAAACACGCGGTTGCTCTTGAAGTGTGCGCCAAAGTCCGGCTACACTGGAAGGACAGATT

[0163] TGGTTGCTGTGCTCTGCGAAAGCCAGTTACCACGGTTAAGCAGTTCCCCAACTGACTTAACCTTCGATCAAACCACCTC

[0164] CCCAGGTGGTTTTTTCGTTTACAGGGCAAAAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTC

[0165] TACTGAACCGCTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTG

[0166] TAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGA

[0167] TCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG

[0168] TGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC

[0169] ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTT

[0170] GCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCC

[0171] CACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTG

[0172] GCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGT

[0173] CGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGA

[0174] GACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTA

[0175] CCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGC

[0176] AGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAG

[0177] ATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCG

[0178] GCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACG

[0179] ACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCG

[0180] CGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACA

[0181] TCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGG

[0182] TTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAATTAATACGACTCA

[0183] CTATA

[0184] SEQ ID NO.6(pET28 vector):

[0185] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT

[0186] GCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTC

[0187] TAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGG

[0188] TTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTC

[0189] TTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCT

[0190] ATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGG

[0191] CACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAAT

[0192] TAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGA

[0193] AAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGA

[0194] TTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATG

[0195] AGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGC

[0196] TCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC

[0197] TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACC

[0198] TGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGA

[0199] GTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACAT

[0200] CATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGC

[0201] ACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTA

[0202] GAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTC

[0203] ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG

[0204] ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA

[0205] AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG

[0206] TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTG

[0207] CCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAAC

[0208] GGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA

[0209] AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGG

[0210] AGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTG

[0211] ATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCT

[0212] TTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG

[0213] CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCT

[0214] TACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCC

[0215] AGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA

[0216] CGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCAC

[0217] CGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTG

[0218] TTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCG

[0219] GTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAG

[0220] AGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGT

[0221] ATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAG

[0222] GGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACG

[0223] AAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTC

[0224] GCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATC

[0225] ATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGA

[0226] AGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTC

[0227] CTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCG

[0228] ACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTG

[0229] CCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCT

[0230] GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA

[0231] GACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGC

[0232] AGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCG

[0233] AGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAG

[0234] CATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCC

[0235] CGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAAC

[0236] TTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTC

[0237] ATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCT

[0238] TCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCA

[0239] CCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGA

[0240] TTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTG

[0241] CCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCG

[0242] CAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAA

[0243] CGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGC

[0244] CATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGG

[0245] CCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCA

[0246] CCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATA

[0247] GGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCG

[0248] CGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAA

[0249] GAAGGAGATATACCATGGgccatcatcatcatcatcacagcggcgaaaacctgtattttcagggcgctagcGGATCCGG

[0250] CTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACC

[0251] CGAAGTATGTCAAAAAGAGGTATGCTatgaagcagcgtattacagtgacaGTTGACAGCGACAGCTATCAGTTGCTCAA

[0252] GGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCT

[0253] GGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGctcttttgctgacgagaacag

[0254] ggGCTGGTGAAatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtga

[0255] tattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaa

[0256] ctttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgtta

[0257] tcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatata

[0258] aATGTCAGGCTCCCTTATACACAGCCAGTCTGCAGGAATTCCCGACCCATCATCAACGGCGAGGAGGGAATTACCATAC

[0259] TGAAACTGTCTCCCAAGACAAGTGTTTTGAACATAGCCGCCGTGGAACAGGATCATCGTGGGGTCTTCAAGTGCATAGC

[0260] CGAAAATAAGGCAGGGGTCAAGTTTCACAACATCGGAGCTGAAAGTCAACTGAAAGCTTGCGGCCGCACTCGAGCACCAC

[0261] CACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAAC

[0262] TAGCATAAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

[0263] Example 1: HNH cytotoxicity experiment and its toxicity inhibition mechanism

[0264] During protein expression, we inserted the target gene of HNH protein (SEQ ID NO.1) between the BamHI / HindIII restriction sites of the pET28 vector (SEQ ID NO.6) to construct a plasmid expressing HNH protein. The plasmid was then transformed into *E. coli* BL21(DE3) bacteria for expression. Normal bacterial plaques grew on the plate, and the OD of the bacteria during single-clone culture was [not specified]. 600 The pH value can be cultured normally to 0.6-0.8. However, once IPTG is added to induce expression, the bacterial culture gradually becomes clear from turbid. As shown in the figure: the bacteria in the centrifuge tube on the left became clear after 1 hour of induction with 0.2 mM IPTG, while the uninduced bacteria on the right remained turbid after 1 hour (see figure). Figure 7 ).

[0265] The Retron system functions as a whole in E. coli. Since the HNH protein may be cytotoxic, we hypothesized that if the intact Retron-Eco4 system is non-toxic under normal physiological conditions, then a component within the system might inhibit HNH toxicity. To verify this hypothesis, we conducted single-component deletion mutation and bacterial toxicity experiments.

[0266] To identify the components in the Retrotron-Eco4 system that inhibit HNH toxicity, different primers were designed (as shown in Table 1), and plasmids containing the complete Retrotron-Eco4 components and single-component deletion plasmids (ΔRT, ΔATPase, ΔHNH) were constructed, such as... Figure 1 The results showed that all genes were cloned into the pCDFDuet vector and then uniformly transformed into BL21(DE3) bacteria for cytotoxicity testing.

[0267] Given that the effector protein ADP-ribosyltransferase in the Retron-Sen2 system is a cryotoxic protein, and that the effector protein ADP-ribosyltransferase in Retron-Eco9 exhibits cytotoxicity at both 37°C and low-temperature conditions (below 25°C), this study investigated the toxicity of HNH at different temperatures of 37°C and 18°C. Furthermore, since the inducer used in the experiment was IPTG, which itself is toxic to bacterial growth, to eliminate this influence, this experiment referenced the experimental method of the Retron-Sen2 system described in the 2022 Nature paper (Jacob Bobonis et al, Bacterial retrons encodephage-defending tripartite toxin–antitoxin systems, 2022, Nature), setting up a no-induction group (no IPTG), a low-concentration IPTG group (0.1 mM), and a high-concentration IPTG group (1 mM). Each experimental condition was performed in triplicate.

[0268] Table 1: Primers for Systematic Single-Component Deletion Mutations

[0269]

[0270] Experimental results Figure 2 -A showed that: (1) In E. coli containing the HNH plasmid, the number of plaques in the low-concentration IPTG group and the high-concentration IPTG group was significantly reduced compared to the uninduced group, and the morphology was also uneven; (2) Bacteria containing the complete Retrotron-Eco4 system plasmid grew well in the uninduced group and the low-concentration IPTG group, but grew poorly in the high-IPTG group due to the toxicity of IPTG; (3) However, it could still be observed that the number of bacterial plaques in the high-IPTG group of HNH was less than that in the high-IPTG group of Retrotron-Eco4, which ruled out the toxic effect of high IPTG concentration on bacteria. Therefore, the experimental results showed that HNH was cytotoxic under different temperature conditions, while the complete Retrotron-Eco4 system was not cytotoxic under different temperature conditions.

[0271] Figure 2 -B bacteria containing ΔRT and ΔHNH plasmids showed different plaque numbers under different temperature conditions after low IPTG induction. Figure 2 Compared to the intact Retron-Eco4 system (IPTG low) plaques in -A, there was no significant cytotoxicity. Figure 2In the -A group, the number of plaques containing HNH alone was high (low IPTG), a consistent trend observed in the high IPTG group. However, bacteria containing the ΔATPase plasmid exhibited varying plaque sizes under different temperature conditions, with significantly fewer plaques after high-IPTG induction compared to the intact Retron-Eco4 system. Therefore, it can be concluded that in the intact system, the toxic effects of HNH are released when ATPase is absent, which can be interpreted as ATPase inhibiting the toxicity of HNH.

[0272] In summary, the cytotoxicity study results indicate that the intact Retron-Eco4 system is non-toxic, while HNH expression alone exhibits cytotoxicity at different temperatures. ATPase also shows an inhibitory effect on HNH toxicity at different temperatures, suggesting that ATPase and HNH may interact under physiological conditions. This further suggests that co-expression of ATPase and HNH is an important way to obtain soluble HNH protein.

[0273] Example 2: HNH interacts with ATPase

[0274] This project first constructed the pCDFDuet-ATPase-FLAG-HNH-HA expression plasmid, such as Figure 3 As shown in Figure -A, a FLAG tag was added to the C-terminus of ATPase, and an HA tag was added to the C-terminus of the HA protein. The constructed plasmid was then transformed into *E. coli* BL21(DE3) competent cells. Expression was induced in 40-50 mL of bacterial culture with 0.2 mM IPTG. The bacterial cells were collected, sonicated on ice, and then centrifuged at low temperature to obtain a supernatant containing soluble ATPase-FLAG and HNH-HA proteins. Subsequently, FLAG-tagged immunomagnetic beads were used to adsorb ATPase-FLAG protein, and the interaction between ATPase and HNH proteins under physiological conditions was determined by analyzing whether ATPase-FLAG protein could bind to and pull down HNH-HA protein.

[0275] Figure 3 SDS-PAGE analysis of -A showed that the eluted FLAG immunomagnetic beads contained protein bands of theoretical size for ATPase-FLAG and HNH-HA, which were bound to the FLAG immunomagnetic beads. Figure 3-B showed that FLAG-tagged proteins precipitated in both the lysis supernatant and the elution sample, indicating the objective presence and effective precipitation of ATPase-FLAG protein in the sample. Subsequent HA precipitation by cutting the theoretical positions of HNH-HA protein on the same membrane revealed that the FLAG immunomagnetic bead eluted sample contained both ATPase and HNH-HA, suggesting a physiological interaction between ATPase and HNH. However, no HNH-HA band was observed in the original lysis buffer, presumably due to the low initial HNH content, with ATPase affinity acting as an enrichment process. In summary, this experiment is consistent with the aforementioned bacterial toxicity and co-expression experiments, demonstrating the binding ability of ATPase to HNH and providing a foundation for further optimization of HNH protein expression and purification methods.

[0276] Example 3: Optimization of HNH protein expression and purification methods

[0277] First, two co-expression plasmids, pCDFDuet-6×His-ATPase-HNH and pCDFDuet-ATPase-HNH-6×His, were constructed. This step involves attaching affinity tags to the ATPase protein and the HNH protein, respectively, to double-validate their interactions. The specific steps for protein expression are as follows:

[0278] 1) Plasmid construction. Two co-expression plasmids, pCDFDuet-6×His-ATPase-HNH and pCDFDuet-ATPase-HNH-6×His, were constructed. The specific sequences of 6×His-ATPase-HNH and ATPase-HNH-6×His are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. The target sequence was inserted between the BamHI / HindIII restriction sites following the first T7 promoter of the pCDFDuet vector.

[0279] 2) Plasmid transformation. Approximately 100 ng of plasmid was transformed into E. coli BL21(DE3) competent cells. The cells were immediately placed on ice and incubated for 30 min, followed by heat shock at 42°C for 60-90 s, and then placed on ice for 2-3 min. Subsequently, 400 μL of antibiotic-free LB was added to the cells, mixed well, and then placed in a shaker for 1 h at 37°C and 200 rpm for recovery and culture. The culture was centrifuged at 3000 rpm for 5 min, 350 μL of LB was discarded, and the cell pellet was mixed with the remaining culture medium and evenly spread onto agar plates corresponding to the plasmid resistance (streptomycin 50 μg / mL). The plates were inverted and incubated overnight at 37°C.

[0280] 3) Small-scale culture. In this project, single clones were selected and cultured in LB broth with the appropriate antibiotic resistance (streptomycin 50 μg / mL) at 37°C and 220 rpm on a shaker until the *E. coli* OD reached its maximum.600 Once the value reaches between 0.6 and 0.8, add IPTG at a final concentration of 0.2 mM and induce expression at 18°C ​​for 12-16 h.

[0281] like Figure 4 As shown, the difference lies in attaching affinity tags to the ATPase and HNH proteins respectively, to cross-validate their interaction and compare enrichment effects. The constructed plasmids were transformed into competent BL21(DE3) cells, followed by E. coli culture and protein expression. The expressed proteins were detected by nickel affinity chromatography SDS-PAGE electrophoresis.

[0282] Experimental results are as follows Figure 5 As shown in -A, when the His6 tag is attached to the C-terminus of the HNH protein, a small amount of expressed HNH protein binds to a corresponding small amount of ATPase protein. When the His6 tag is attached to the N-terminus of the ATPase protein, a large amount of expressed ATPase protein can carry out a small amount of expressed HNH protein. Figure 5 -B). The protein expression results of the two expression plasmids showed that the tag band pulled down more HNH protein from the ATPase protein (6L His6-ATPase bacteria could obtain about 2-3 mg HNH, and 6L His6-HNH bacteria could obtain about 1-2 mg HNH). Therefore, the pCDFDuet-His6-ATPase-HNH plasmid was subsequently selected for HNH protein expression.

[0283] Subsequently, we used Superdex to purify the Ni-NTA product. TM Further purification was performed using 200 mL gel filtration chromatography. As expected, the initial purified product yielded a homogeneous and stable complex of ATPase and HNH upon gel filtration chromatography. The molecular weight of the ATPase protein was approximately 62.8 kDa, and the molecular weight of the HNH protein was approximately 30.2 kDa, with a theoretical peak position between 11 and 12 mL. The results are as follows... Figure 5As shown in Figure -C, the peak position of "Peak 1" is between 11-12 mL. The theoretical molecular weight of the protein eluting at this position is approximately 400 kDa. Combined with the SDS-PAGE results, it is confirmed that "Peak 1" contains only ATPase protein, therefore it is speculated that "Peak 1" is a hexameric or heptamer of ATPase. "Peak 2" is the most dominant and symmetrical protein, with a peak position between 13-14 mL. The theoretical molecular weight of the protein eluting at this position is approximately 158 kDa. The SDS-PAGE results show that "Peak 2" contains only ATPase protein, therefore "Peak 2" is a dimerized ATPase. The peak position of "Peak 3" is between 16-17 mL. The theoretical molecular weight of the protein eluting at this position is approximately 29 kDa. The SDS-PAGE results show that "Peak 3" is mainly a monomeric HNH protein.

[0284] Co-expression experiments showed that ATPase directly binds to HNH, but the binding is unstable or weak, leading to separation during molecular sieve chromatography. It is speculated that this is because ATP is unstable and is continuously consumed during protein purification. The conformation of ATPase changes due to ATP hydrolysis, causing it to separate from HNH and ultimately yielding the individual HNH protein. This expression and purification method successfully obtained a single HNH protein, laying the foundation for subsequent studies on HNH enzyme activity.

[0285] Example 4 Enzyme activity detection of purified HNH

[0286] The genomes of bacterial hosts and bacteriophages are supercoiled. Therefore, we used the supercoiled pUC19 plasmid as a cleavage substrate to detect whether HNH (purified protein) possesses endonuclease activity and whether it cleaves single-stranded or double-stranded DNA. The experiment consisted of three groups: supercoiled plasmid (extracted pUC19), linear plasmid (pUC19 plasmid treated with 50U of commercial EcoRI high-fidelity enzyme, in the linear state), and Nicked plasmid (pUC19 treated with 50U of commercial BspQI enzyme, in the Nicked state with a single-stranded notch), corresponding to the three scenarios: no endonuclease activity of HNH, HNH cleaving of double-stranded DNA, and HNH cleaving of single-stranded DNA, respectively. The Nicked plasmid was eventually released into an open circular state.

[0287] Experimental results are as follows Figure 6As shown in Figure A, in agarose gel electrophoresis, the supercoiled pUC19 band has the fastest electrophoretic speed due to its compact structure. The linearized pUC19 band is located in the middle, while the loosely structured, open-ended pUC19 band has the slowest electrophoretic speed and is located at the top. HNH can cleave 200 ng of supercoiled pUC19 into the nickel state at a protein concentration of 100 nM, and at a concentration of 1 μM, it can almost completely cleave 200 ng of pUC19, producing linear cleavage products, but mainly nickel-state cleavage products. This indicates that HNH possesses nickase activity for cleaving single-stranded DNA.

[0288] It is known that the activity of HNH endonucleases is supported by the divalent metal ion Mg. 2+ Due to the dependence of HNH, the effects of some common divalent metal ions on the nicking enzyme activity of HNH were then tested. The control group consisted of untreated pUC19 and a reaction system with the addition of the metal ion chelating agent EDTA. The experimental group contained 1 mM of metal ions. The results are as follows: Figure 6 -B shows the removal of Zn 2+ In addition, Mg 2+ Mn 2 + , Ca 2+ Ni 2+ Both can promote the cleavage activity of HNH.

[0289] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for expressing and purifying HNH toxic protein, wherein the method is non-toxic to the host, comprising the following steps: 1) The HNH nuclease coding frame and the ATPase coding frame were simultaneously and independently expressed in the same cell. The expression of the HNH nuclease and ATPase was induced by IPTG. The ATPase coding frame encodes the amino acid sequence shown in SEQ ID NO.

2. The amino acid encoded by the HNH nuclease coding frame is as follows: MILKRINKTAEDQFLINFKAQNPNGTWDEFRNHEQGILYKRLKQHICNDQMYLCAYCEIDLDRENEHEIKVEHFKSKSGSLPGGSNWHLEWSNLLAVCLGGTNTGDDFELPANLSCDSYKSHYEDKNKINDKDWTGKILLPLTLPDAHNFFTFEKVTGKLLPNESYCNTISIDGKPAAETLSIVTKTIEVLNLNCSRLNNARRKLLFHFNNCARERNLRKLHNLLLQWNQGEPKFFQTTRDIIIRDDRICQGLLNGTIRY; 2) Preliminary purification of the expression product; 3) The initially purified product is further purified; in, The host is Escherichia coli, and in step 1), the HNH nuclease coding frame and the ATPase coding frame are expressed independently and simultaneously in the same vector.

2. The method for expressing and purifying HNH toxic protein according to claim 1, characterized in that, In step 1), an upstream tag protein coding frame is connected to the ATPase coding frame, and the tag protein coding frame and the ATPase coding frame together express a fusion protein containing the tag protein and ATPase.

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