CN118240856A

CN118240856A - Escherichia coli toxic protein and application and prokaryotic expression method thereof

Info

Publication number: CN118240856A
Application number: CN202410524229.9A
Authority: CN
Inventors: 杨泽婧; 胡长江; 范婷
Original assignee: Second Affiliated Hospital Army Medical University
Current assignee: Second Affiliated Hospital Army Medical University
Priority date: 2024-04-29
Filing date: 2024-04-29
Publication date: 2024-06-25

Abstract

The invention provides a high-efficiency expression purification method of a cytotoxic protein HNH with endonuclease activity, and proves the endonuclease activity of HNH. The implementation of the invention not only optimizes the expression and purification method of HNH, but also provides a potential tool enzyme.

Description

Escherichia coli toxic protein and application and prokaryotic expression method thereof

Field of application

The invention relates to the technical field of enzyme and protein expression and purification methods, in particular to an expression and purification method of toxic protein HNH with endonuclease activity.

Background

HNH enzymes, also known as "ββα -me" endonucleases, all members of this family have a highly similar catalytic motif- ββα topology consisting of 30 to 40 amino acids responsible for binding and cleavage of nucleic acids, and are structurally characterized by the folding of three conserved amino acid His-Asn-His residues (H-N-H) into two antiparallel β sheets, an α helix and a β - α -metal topology that binds divalent metal cations, ultimately folding into ββα -metal topology.

The sequence conservation of HNH proteins is manifested by His being conserved at the end of the β1 chain (corresponding to the first letter "H" in HNH), metal ions binding to highly conserved Asn/His residues located within the α -helix (corresponding to the last letter "H" in HNH), conserved Asn at the beginning of the β2 chain (corresponding to the letter "N" in HNH), which typically hydrogen bonds with the backbone atoms next to catalytic His, thus playing an important structural role in fixing the position and orientation of the general bases. When the HNH nuclease functions as a monomer, a single gap is made in the nucleic acid to degrade the exogenous or host genome (Nickase endonuclease), and when functioning as a homodimer, double-stranded DNA breaks are introduced for DNA restriction, integration, recombination, and repair.

Earlier studies show that when HNH protein is expressed by the prokaryote, the experimental phenomenon is that the escherichia coli can be cultured, but once the escherichia coli is expressed by induction, bacterial liquid becomes clear gradually, and cytotoxicity of the HNH protein is estimated initially. Thus, it is a key to improve the expression efficiency of HNH protein to build an expression purification system which does not generate cytotoxicity. In addition, the HNH protein family has a plurality of different sequences, and the identification of the novel HNH protein with endonuclease activity can further enrich the number of tool enzymes, so that more choices are provided for the modification and optimization of the tool enzymes.

Disclosure of Invention

Aiming at the problems existing in the prior art, the embodiment of the invention provides a method for coexpression of HNH protein and ATPase protein in the same open reading frame and obtaining soluble target HNH protein.

The primary object of the present invention is to provide an HNH toxic protein expression vector, which comprises an ATPase coding frame and an HNH coding frame, and the ATPase coding frame and the HNH coding frame can be simultaneously and independently expressed.

Further, the HNH toxic protein expression vector is formed by inserting an ATPase coding frame and an HNH coding frame into any prokaryotic expression vector.

Further, the HNH toxic protein expression vector is formed by inserting ATPase coding frames and HNH coding frames into the positions of the expressible ATPase coding frames and HNH coding frames of any prokaryotic expression vector.

Further, the ATPase coding frame and the HNH coding frame are respectively from an Escherichia coli Retron-Eco4 system.

Further, the ATPase coding box and the HNH coding box are from the same bacterium Retron-Eco4 system.

Further, the ATPase coding frame and the HNH coding frame are from the same strain of bacteria Retron-Eco4 system.

Further, the ATPase-encoded protein and HNH-encoded protein have interactions, and the ATPase-encoded protein inhibits cytotoxicity of the HNH-encoded protein.

Further, the ATPase-encoding and HNH-encoding proteins have unstable interactions or weak binding forces, which separate during molecular sieve chromatography.

In a preferred embodiment, the ATPase coding box or HNH coding box is linked to a tag protein coding box which together with the ATPase coding box or HNH coding box expresses a fusion protein comprising the tag protein and ATPase or HNH, said tag protein being located 5 'or 3' to the ATPase coding box or HNH coding box.

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

In a preferred embodiment, the HNH coding box is located downstream of the ATPase coding box, and a spacer sequence is included between the two, which allows independent expression of the HNH coding box and the ATPase coding box.

In a preferred embodiment, the HNH coding box, ATPase coding box, spacer sequence and tag protein coding box are formed as follows: F-ATPase-B-HNH or ATPase-B-HNH-F, wherein F is the coding box of the tag protein, B is the spacer sequence, preferably F is 6XHis, and B is RBS (ribosome binding site).

In a preferred embodiment, the ATPase coding box encodes an amino acid sequence as shown in SEQ ID no2 and the HNH coding box expresses an amino acid sequence as shown in SEQ ID no1.

In a preferred embodiment, the HNH toxic protein expression vector comprises the HNH coding box, the ATPase coding box, the spacer sequence and the tag protein coding box having the structure as shown in SEQ ID NO.3 or SEQ ID NO.4, preferably SEQ ID NO.3.

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

A method for purifying HNH toxic protein expression, the method comprising the steps of:

1) The HNH coding box and the ATPase coding box are simultaneously and independently expressed in the same cell.

2) And (5) primarily purifying the expression product.

3) The initially purified product was further purified.

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

Further, the ATPase coding box and the HNH coding box in the step 1) are ATPase coding box and HNH coding box from the E.coli Retron-Eco4 system.

Further, the ATPase coding box and the HNH coding box in the step 1) are both ATPase coding box and HNH coding box from the same bacteria Retron-Eco4 system.

Further, the ATPase coding box and the HNH coding box in the step 1) are both ATPase coding box and HNH coding box from the same strain of bacteria Retron-Eco4 system.

Further, the ATPase-encoding box-encoded protein and HNH-encoding box-encoded protein in step 1) have interactions, and the ATPase-encoding box-encoded protein can inhibit cytotoxicity of the HNH-encoding box-encoded protein.

Further, the protein encoded by the ATPase coding box and the protein encoded by the HNH coding box in step 1) have unstable interactions or weak binding forces, and can be separated during molecular sieve chromatography.

Further, in step 1), the ATPase coding box or HNH coding box is linked to a tag protein coding box, which together with the ATPase coding box or HNH coding box expresses a fusion protein comprising the tag protein and ATPase or HNH, which tag protein is located at the 5 'or 3' end of the ATPase coding box.

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

In a preferred embodiment, step 3) is performed using gel filtration chromatography.

An endonuclease having the sequence of the amino acid sequence shown in SEQ ID NO. 1.

In a preferred embodiment, the endonuclease has the amino acid sequence shown in SEQ ID NO. 1.

Further, the endonuclease has a nicking enzyme activity that cleaves single-stranded DNA (NICKASE ACTIVITY).

The invention has the following beneficial effects:

1. Provides a novel vector and a purification method for expression and purification of toxic proteins, and improves the expression efficiency of the toxic proteins.

2. A novel enzyme having nicking enzyme activity (NICKASE ACTIVITY) for cleaving single-stranded DNA is provided.

Drawings

The method of the present invention and its advantageous effects will be described in detail below with reference to the accompanying drawings and detailed description.

Fig. 1: the expression plasmid required for cytotoxicity experiment was constructed.

Fig. 2: cytotoxicity test results. The bacterial concentration values gradually decreased from top to bottom, and the images shown are representative of three replicates. (a) HNH is cytotoxic; (B) ATPase inhibits HNH cytotoxicity.

Fig. 3: co-IP experimental results of ATPase and HNH. (A) The detection result of the SDS-PAGE of the co-expression of ATPase-FLAG and HNH-HA; (B) the results of ATPase-FLAG and HNH-HA WESTERN blot.

FIG. 4. Construction of ATPase and HNH co-expression plasmids.

Fig. 5: results of ATPase co-expression with HNH. (A) Co-expression nickel affinity chromatography SDS-PAGE results of the tag band on HNH; (B) Co-expression nickel affinity chromatography SDS-PAGE results of the tag band on ATPase; (C) The result of the purification of the product Superdex200 gel filtration chromatography of the co-expression product of ATPase and HNH is obtained.

Fig. 6: HNH endoenzyme activity results. (A) the presence of Nickase enzyme activities in 100nM HNH protein; (B) effect of different divalent cations on HNH enzyme activity; (C) effect of different nucleotides on HNH enzyme activity; (D) schematic of endonuclease experiment.

Fig. 7: IPTG is added to induce expression, and bacterial liquid is changed. Bacteria became clear after 1h of induction with 0.2mM IPTG added to the left centrifuge tube, and remained cloudy after 1h of non-induced bacteria on the right.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

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

SEQ ID NO.1(HNH)：

MILKRINKTAEDQFLINFKAQNPNGTWDEFRNHEQGILYKRLKQHICNDQMYLCAYCEIDLDRENEHEIKVEHFKSKSG

SLPGGSNWHLEWSNLLAVCLGGTNTGDDFELPANLSCDSYKSHYEDKNKINDKDWTGKILLPLTLPDAHNFFTFEKVTG

KLLPNESYCNTISIDGKPAAETLSIVTKTIEVLNLNCSRLNNARRKLLFHFNNCARERNLRKLHNLLLQWNQGEPKFFQ

TTRDIIIRDDRICQGLLNGTIRYYPYDVPDYA

SEQ ID NO.2(ATPase)：

MEQNLPSRITKLIKKSESGDFASSYQLYKVFGSKEYGVEPDEKMSDYFKELSAKQLEGGQLRVADIHLENYKGFESLIM

DFSMKKNSTILVGNNGCGKSTILDAIQKGLTHLSSRLSTRSHNGDGIEKHELRKGQNYASIAINYDYMGIRFPMIIATT

EPGYEDRAKSNYSGINELGSIFKTAHSINPNVSFPLIAMYTVERANDVSTRDIENSEEIKEAQIWDKFKAYNKSLTGKA

DFKLFFRWFKELIEIENSDNADITALRAEIRAKEKDLDNPLLKALLAENKNSETTKKLLEDHQNSLKVLKEKLNSYYSV

NSKTLHTVEDAMYSFLPGFSNLKLQRAPLDLIVDKNNVSLSVLQLSQGEKTILALIADIARRLTLLNPNSVNPLDGTGI

VLIDEIDLHLHPSWQQNIIPRLEKTFKNIQFIVTTHSPQVCHTIDSQNIWLLKNGQKFKAPKGVRGAISSWVLENLFEV

AQRPPEDKYTKLLQEYKNLVFSEKYASEDARKLGATLSQHFGPDDETLVELKLEIEKRIWEDDFEKDQDYKDDDDKSEQ ID NO.3(6XHis-ATPase-RBS-HNH)：

catcatcatcatcatcacagcggcgaaaacctgtattttcagggcgctagcGGATCCATGGAACAGAACTTACCGAGTA

GAATAACTAAATTAATTAAAAAATCGGAAAGCGGCGATTTTGCTTCTTCGTATCAGCTTTATAAAGTATTTGGCTCGAA

AGAGTACGGAGTTGAGCCAGATGAAAAAATGTCTGATTACTTCAAAGAATTATCAGCTAAACAGTTAGAAGGTGGTCAA

CTGAGAGTTGCTGATATTCATTTGGAAAACTACAAAGGGTTTGAGTCCCTAATAATGGATTTTTCCATGAAAAAAAACT

CTACAATTTTAGTAGGAAATAATGGCTGTGGGAAAtcgacGATTCTCGATGCAATCCAGAAGGGGTTAACACATCTATC

CTCGAGATTATCTACTCGCTCGCATAATGGCGATGGTATCGAAAAACATGAGTTAAGAAAAGGACAAAACTATGCATCG

ATCGCTATAAATTACGACTATATGGGAATACGTTTTCCTATGATCATAGCTACAACTGAACCTGGTTATGAGGATAGAG

CAAAAAGTAACTATAGCGGTATTAATGAGTTAGGAAGCATTTTCAAAACAGCCCATTCAATCAATCCAAATGTTTCATT

TCCTTTAATTGCAATGTATACAGTTGAAAGGGCTAATGACGTTTCTACTAGAGATATTGAAaattcAGAAGAAATTAAA

GAAGCTCAAATCTGGGATAAATTCAAAGCATATAATAAGAGCCTGACAGGAAAGGCTGATTTCAAATTATTCTTCAGAT

GGTTTAAAGAACTAATAGAAATCGAAAACTCTGATAACGCTGATATAACAGCATTAAGAGCAGAGATTCGTGCTAAAGA

AAAAGACTTAGACAATCCATTGCTAAAAGCTCTTCTGGCAGAGAATAAAAATTCTGAGACTACTAAAAAATTGTTAGAA

GATCATCAGAACTCTCTGAAAGTTTTAAAAGAGAAATTAAATAGCTATTATTCAGTCAATAGTAAAACATTACACACTG

TTGAAGATGCAATGTATTCTTTCCTTCCTGGTTTTAGCAACCTTAAACTTCAAAGGGCGCCTCTTGATCTGATAGTGGA

TAAGAATAATGTTTCCTTAAGTGTTCTGCAATTATCTCAAGGTGAAAAAACCATTTTAGCATTAATTGCAGATATTGCT

CGTAGATTGACATTGTTAAACCCTAATAGTGTTAACCCTTTGGACGGTACTGGAATTGTATTAATCGATGAAATAGACC

TCCATTTACATCCATCATGGCAGCAAAATATTATTCCTCGTCTTGAGAAAACGTTTAAGAATATTCAATTTATAGTCAC

GACTCATAGTCCACAAGTTTGTCATACTATTGATAGTCAGAATATATGGTTGTTAAAGAATGGCCAAAAGTTTAAAGCA

CCGAAAGGAGTTAGAGGAGCAATATCTTCTTGGGTACTGGAGAACTTGTTCGAAGTTGCTCAAAGGCCGCCAGAGGATA

AGTACACAAAACTCTTACAGGAATATAAAAATTTAGTATTTTCAGAAAAATATGCTAGTGAAGATGCAAGAAAGCTAGG

TGCTACTTTATCCCAACATTTCGGACCGGATGATGAAACCTTAGTTGAGTTAaagctAGAAATTGAAAAAAGAATTTGG

GAGGATGATTTTGAAAAGGATCAAGATTACAAGGACGACGATGACAAGTAAgaattcaatcaataggagaaatcaatGG

ATCCATGATTTTGAAAAGGATCAATAAAACTGCTGAAGATCAATTCTTAATTAATTTTAAAGCTCAAAATCCAAATGGG

ACTTGGGATGAATTTAGGAATCATGAACAAGGTATTTTATATAAGAGGTTAAAGCAACATATTTGCAATGATCAAATGT

ACCTTTGTGCGTATTGTGAGATAGATTTAGATCGAGAAAATGAACATGAAATAAAAGTAGAGCATTTCAAATCTAAATC

TGGTTCGCTCCCTGGTGGAAGTAACTGGCATTTAGAGTGGTCTAATCTCTTAGCTGTATGCCTAGGAGGTACAAATACA

GGTGATGATTTCGAATTACCAGCTAATCTAAGTTGTGATTCATATAAGTCACATTATGAAGACAAAAATAAAATCAATG

ATAAAGACTGGACAGGCAAAATCCTGTTACCTTTAACGCTTCCGGATGCACACAATTTTTTTACTTTCGAGAAAGTTAC

AGGTAAGTTGCTACCTAATGAATCATACTGTAATACTATTAGCATAGATGGTAAACCTGCTGCAGAAACACTAAGTATT

GTAACTAAAACAATAGAAGTTCTAAATTTAAACTGCAGCAGGCTAAATAATGCCAGAAGAAAACTGCTGTTTCACTTTA

ATAATTGCGCACGTGAAAGAAACTTGAGAAAGCTCCATAATCTATTATTACAATGGAATCAAGGTGAGCCTAAATTTTT

CCAAACGACACGAGATATAATAATTCGTGATGATAGAATCTGCCAAGGGTTACTGAACGGAACGATAAGATATTACCCA

TACGATGTTCCAGATTACGCTTAA

SEQ ID NO.4(ATPase-HNH-6×His)：

ATGGAACAGAACTTACCGAGTAGAATAACTAAATTAATTAAAAAATCGGAAAGCGGCGATTTTGCTTCTTCGTATCAGC

TTTATAAAGTATTTGGCTCGAAAGAGTACGGAGTTGAGCCAGATGAAAAAATGTCTGATTACTTCAAAGAATTATCAGC

TAAACAGTTAGAAGGTGGTCAACTGAGAGTTGCTGATATTCATTTGGAAAACTACAAAGGGTTTGAGTCCCTAATAATG

GATTTTTCCATGAAAAAAAACTCTACAATTTTAGTAGGAAATAATGGCTGTGGGAAAtcgacGATTCTCGATGCAATCC

AGAAGGGGTTAACACATCTATCCTCGAGATTATCTACTCGCTCGCATAATGGCGATGGTATCGAAAAACATGAGTTAAG

AAAAGGACAAAACTATGCATCGATCGCTATAAATTACGACTATATGGGAATACGTTTTCCTATGATCATAGCTACAACT

GAACCTGGTTATGAGGATAGAGCAAAAAGTAACTATAGCGGTATTAATGAGTTAGGAAGCATTTTCAAAACAGCCCATT

CAATCAATCCAAATGTTTCATTTCCTTTAATTGCAATGTATACAGTTGAAAGGGCTAATGACGTTTCTACTAGAGATAT

TGAAaattcAGAAGAAATTAAAGAAGCTCAAATCTGGGATAAATTCAAAGCATATAATAAGAGCCTGACAGGAAAGGCT

GATTTCAAATTATTCTTCAGATGGTTTAAAGAACTAATAGAAATCGAAAACTCTGATAACGCTGATATAACAGCATTAA

GAGCAGAGATTCGTGCTAAAGAAAAAGACTTAGACAATCCATTGCTAAAAGCTCTTCTGGCAGAGAATAAAAATTCTGA

GACTACTAAAAAATTGTTAGAAGATCATCAGAACTCTCTGAAAGTTTTAAAAGAGAAATTAAATAGCTATTATTCAGTC

AATAGTAAAACATTACACACTGTTGAAGATGCAATGTATTCTTTCCTTCCTGGTTTTAGCAACCTTAAACTTCAAAGGG

CGCCTCTTGATCTGATAGTGGATAAGAATAATGTTTCCTTAAGTGTTCTGCAATTATCTCAAGGTGAAAAAACCATTTT

AGCATTAATTGCAGATATTGCTCGTAGATTGACATTGTTAAACCCTAATAGTGTTAACCCTTTGGACGGTACTGGAATT

GTATTAATCGATGAAATAGACCTCCATTTACATCCATCATGGCAGCAAAATATTATTCCTCGTCTTGAGAAAACGTTTA

AGAATATTCAATTTATAGTCACGACTCATAGTCCACAAGTTTGTCATACTATTGATAGTCAGAATATATGGTTGTTAAA

GAATGGCCAAAAGTTTAAAGCACCGAAAGGAGTTAGAGGAGCAATATCTTCTTGGGTACTGGAGAACTTGTTCGAAGTT

GCTCAAAGGCCGCCAGAGGATAAGTACACAAAACTCTTACAGGAATATAAAAATTTAGTATTTTCAGAAAAATATGCTA

GTGAAGATGCAAGAAAGCTAGGTGCTACTTTATCCCAACATTTCGGACCGGATGATGAAACCTTAGTTGAGTTAaagct

AGAAATTGAAAAAAGAATTTGGGAGGATGATTTTGAAAAGGATCAAGATTACAAGGACGACGATGACAAGTAAgaattc

aatcaataggagaaatcaatGGATCCATGATTTTGAAAAGGATCAATAAAACTGCTGAAGATCAATTCTTAATTAATTT

TAAAGCTCAAAATCCAAATGGGACTTGGGATGAATTTAGGAATCATGAACAAGGTATTTTATATAAGAGGTTAAAGCAA

CATATTTGCAATGATCAAATGTACCTTTGTGCGTATTGTGAGATAGATTTAGATCGAGAAAATGAACATGAAATAAAAG

TAGAGCATTTCAAATCTAAATCTGGTTCGCTCCCTGGTGGAAGTAACTGGCATTTAGAGTGGTCTAATCTCTTAGCTGT

ATGCCTAGGAGGTACAAATACAGGTGATGATTTCGAATTACCAGCTAATCTAAGTTGTGATTCATATAAGTCACATTAT

GAAGACAAAAATAAAATCAATGATAAAGACTGGACAGGCAAAATCCTGTTACCTTTAACGCTTCCGGATGCACACAATT

TTTTTACTTTCGAGAAAGTTACAGGTAAGTTGCTACCTAATGAATCATACTGTAATACTATTAGCATAGATGGTAAACC

TGCTGCAGAAACACTAAGTATTGTAACTAAAACAATAGAAGTTCTAAATTTAAACTGCAGCAGGCTAAATAATGCCAGA

AGAAAACTGCTGTTTCACTTTAATAATTGCGCACGTGAAAGAAACTTGAGAAAGCTCCATAATCTATTATTACAATGGA

ATCAAGGTGAGCCTAAATTTTTCCAAACGACACGAGATATAATAATTCGTGATGATAGAATCTGCCAAGGGTTACTGAA

CGGAACGATAAGATATCACCACCACCACCACCACTAA

SEQ ID NO.5(pCDFDuet vector)：

GGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGAGATATACCATGGGCAGC

AGCAGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAG

AAAGTAATCGTATTGTACACGGCCGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATT

CCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGGCAGATCTCAATTGGATATCGGCCGGCCACGC

GATCGCTGACGTCGGTACCCTCGAGTCTGGTTCTACTAGCGCAGCTTAATTAACCTAGGCTGCTGCCACCGCTGAGCAA

TAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAACCTCAGGCATTTGAGAAGCACA

CGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGA

ACCGACGACCGGGTCATCGTGGCCGGATCTTGCGGCCCCTCGGCTTGAACGAATTGTTAGACATTATTTGCCGACTACC

TTGGTGATCTCGCCTTTCACGTAGTGGACAAATTCTTCCAACTGATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTC

CAAGATAAGCCTGTCTAGCTTCAAGTATGACGGGCTGATACTGGGCCGGCAGGCGCTCCATTGCCCAGTCGGCAGCGAC

ATCCTTCGGCGCGATTTTGCCGGTTACTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGCTCATCGCCA

GCCCAGTCGGGCGGCGAGTTCCATAGCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAA

AGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTTTGTCAGCAAGATAGCCAGATCAATGTC

GATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCATTGCGCTGCCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGA

TAACGCCACGGAATGATGTCGTCGTGCACAACAATGGTGACTTCTACAGCGCGGAGAATCTCGCTCTCTCCAGGGGAAG

CCGAAGTTTCCAAAAGGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGCAAATC

AATATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAATGTACGGCCAGCAACGTCGGTTCGAGATGG

CGCTCGATGACGCCAACTACCTCTGATAGTTGAGTCGATACTTCGGCGATCACCGCTTCCCTCATACTCTTCCTTTTTC

AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT

AGCTAGCTCACTCGGTCGCTACGCTCCGGGCGTGAGACTGCGGCGGGCGCTGCGGACACATACAAAGTTACCCACAGAT

TCCGTGGATAAGCAGGGGACTAACATGTGAGGCAAAACAGCAGGGCCGCGCCGGTGGCGTTTTTCCATAGGCTCCGCCC

TCCTGCCAGAGTTCACATAAACAGACGCTTTTCCGGTGCATCTGTGGGAGCCGTGAGGCTCAACCATGAATCTGACAGT

ACGGGCGAAACCCGACAGGACTTAAAGATCCCCACCGTTTCCGGCGGGTCGCTCCCTCTTGCGCTCTCCTGTTCCGACC

CTGCCGTTTACCGGATACCTGTTCCGCCTTTCTCCCTTACGGGAAGTGTGGCGCTTTCTCATAGCTCACACACTGGTAT

CTCGGCTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTAAGCAAGAACTCCCCGTTCAGCCCGACTGCTGCGCCTTAT

CCGGTAACTGTTCACTTGAGTCCAACCCGGAAAAGCACGGTAAAACGCCACTGGCAGCAGCCATTGGTAACTGGGAGTT

CGCAGAGGATTTGTTTAGCTAAACACGCGGTTGCTCTTGAAGTGTGCGCCAAAGTCCGGCTACACTGGAAGGACAGATT

TGGTTGCTGTGCTCTGCGAAAGCCAGTTACCACGGTTAAGCAGTTCCCCAACTGACTTAACCTTCGATCAAACCACCTC

CCCAGGTGGTTTTTTCGTTTACAGGGCAAAAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTC

TACTGAACCGCTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTG

TAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGA

TCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG

TGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC

ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTT

GCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCC

CACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTG

GCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGT

CGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGA

GACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTA

CCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGC

AGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAG

ATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCG

GCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACG

ACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCG

CGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACA

TCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGG

TTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAATTAATACGACTCA

CTATA

SEQ ID NO.6(pET28 vector)：

TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT

GCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTC

TAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGG

TTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTC

TTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCT

ATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGG

CACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAAT

TAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGA

AAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGA

TTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATG

AGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGC

TCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC

TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACC

TGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGA

GTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACAT

CATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGC

ACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTA

GAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTC

ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG

ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA

AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG

TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTG

CCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAAC

GGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA

AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGG

AGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTG

ATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCT

TTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG

CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCT

TACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCC

AGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA

CGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCAC

CGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTG

TTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCG

GTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAG

AGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGT

ATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAG

GGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACG

AAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTC

GCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATC

ATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGA

AGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTC

CTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCG

ACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTG

CCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCT

GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA

GACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGC

AGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCG

AGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAG

CATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCC

CGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAAC

TTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTC

ATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCT

TCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCA

CCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGA

TTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTG

CCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCG

CAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAA

CGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGC

CATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGG

CCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCA

CCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATA

GGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCG

CGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAA

GAAGGAGATATACCATGGgccatcatcatcatcatcacagcggcgaaaacctgtattttcagggcgctagcGGATCCGG

CTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACC

CGAAGTATGTCAAAAAGAGGTATGCTatgaagcagcgtattacagtgacaGTTGACAGCGACAGCTATCAGTTGCTCAA

GGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCT

GGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGctcttttgctgacgagaacag

ggGCTGGTGAAatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtga

tattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaa

ctttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgtta

tcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatata

aATGTCAGGCTCCCTTATACACAGCCAGTCTGCAGGAATTCCCGACCCATCATCAACGGCGAGGAGGGAATTACCATAC

TGAAACTGTCTCCCAAGACAAGTGTTTTGAACATAGCCGCCGTGGAACAGGATCATCGTGGGGTCTTCAAGTGCATAGC

CGAAAATAAGGCAGGGTCAAGTTTCACAACATCGGAGCTGAAAGTCAACTGAAAGCTTGCGGCCGCACTCGAGCACCAC

CACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAAC

TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

Example 1HNH cytotoxicity assay and mechanism of toxicity inhibition

In the protein expression process, a target gene of HNH protein (SEQ ID NO. 1) is inserted between BamHI/HindIII cleavage sites of a pET28 vector (SEQ ID NO. 6) to construct a plasmid for expressing HNH protein, the plasmid is transferred into escherichia coli BL21 (DE 3) bacteria for expression, and the fact that normal bacterial plaques can grow on the flat plate is found, and the OD ₆₀₀ value of the bacteria can be normally cultured to 0.6-0.8 when monoclonal culture is selected. However, once IPTG was added to induce expression, the bacterial solution became clear from turbidity. As shown in the figure: bacteria became clear after 1h of induction with 0.2mM IPTG in the left centrifuge tube, and remained cloudy after 1h of non-induced bacteria on the right (see FIG. 7).

The Retron system acts as a whole in E.coli and there may be cytotoxicity of HNH protein, so we speculate that under normal physiological conditions, if there is no toxicity in the Retron-Eco4 complete system, the presence of a certain component in the system inhibits HNH toxicity. To verify this hypothesis, we performed single component deletion mutations and bacterial toxicity experiments.

In order to identify components of the Retron-Eco4 system that inhibit HNH toxicity, different primers were designed (as in Table 1), retron-Eco4 whole-component plasmids and single-component deletion plasmids (ΔRT, ΔATPase, ΔHNH) were constructed, as shown in FIG. 1, all genes were cloned into pCDFDuet vector and transformed into BL21 (DE 3) bacteria in unison for cytotoxicity testing.

Whereas the effector protein ADP-ribosyl transferase in the Retron-Sen2 system is a low temperature toxic toxin protein, the effector protein ADP-ribosyl transferase of Retron-Eco9 is cytotoxic both at 37℃and at low temperature (below 25 ℃). Thus, in this study, it was investigated whether HNH was toxic at a temperature different from that of 18℃at 37 ℃. Furthermore, since the inducer in the experiment was IPTG, there was some toxicity of IPTG to the growth itself of bacteria, and in order to eliminate this effect, the experiment was conducted with reference to the Retron-Sen2 system of the journal (Jacob Bobonis et al,Bacterial retrons encode phage-defending tripartite toxin–antitoxin systems,2022,Nature) of Nature in 2022, and a non-induced group (no IPTG), a low concentration IPTG group (0.1 mM), and a high concentration IPTG group (1 mM) were set. Three replicates were set for each experimental condition.

Table 1: systematic single component deletion mutant primers

Experimental results fig. 2-a shows: (1) Coli containing HNH plasmid, the bacterial plaque of the low concentration IPTG group and the bacterial plaque of the high concentration IPTG group are obviously reduced compared with the bacterial plaque of the uninduced group, and the morphology is also nonuniform; (2) Bacteria containing Retron-Eco4 complete system plasmids grow well in the uninduced group and the low-concentration IPTG group, and grow poorly in the high-IPTG group because of the toxic effect of IPTG; (3) However, it was still observed that the HNH high IPTG group had a smaller number of bacterial plaques than the Retron-Eco4 high IPTG group, where the toxic effects of high IPTG concentrations on the bacteria could be excluded. Thus, the experimental result shows that HNH has cytotoxicity under different temperature conditions, and the complete Retron-Eco4 system has no cytotoxicity under different temperature conditions.

The bacteria containing ΔRT and ΔHNH plasmids in FIG. 2-B were not significantly cytotoxic after low IPTG induction (IPTG low) compared to the plaques of the complete Retron-Eco4 system (IPTG low) in FIG. 2-A, and were more numerous than the plaques of HNH alone (IPTG low) in FIG. 2-A, consistent with the high IPTG group case; however, bacteria containing ΔATPase plasmids exhibited different sizes of bacterial plaques under different temperature conditions, and the bacterial plaques after high IPTG induction were significantly reduced compared with the complete Retron-Eco4 system. It was then concluded that in the complete system, the toxic effects of HNH are released after ATPase deletion, which can be understood as the toxicity of ATPase to inhibit HNH.

In conclusion, the cytotoxicity experimental research results show that the complete Retron-Eco4 system has no toxicity, the independent expression HNH has cytotoxicity at different temperatures, and ATPase has HNH toxicity inhibition effect at different temperatures, which suggests that ATPase and HNH may have interaction under physiological conditions, and further suggests that the co-expression of ATPase and HNH is an important way for obtaining soluble HNH protein.

Example 2 interaction of HNH with ATPase

The project first constructed pCDFDuet-ATPase-FLAG-HNH-HA expression plasmid, as shown in FIG. 3-A, FLAG tag on the C-terminal band of ATPase and HA tag on the C-terminal band of HA protein. And then the constructed plasmid is transformed into escherichia coli BL21 (DE 3) expression competent cells, 0.2mM IPTG induces and expresses 40-50mL bacterial liquid, after bacterial cells are collected, bacterial cells are crushed by ultrasound on ice, and then the supernatant containing soluble ATPase-FLAG and HNH-HA proteins is obtained by low-temperature centrifugation. And then, utilizing the FLAG tag immunomagnetic beads to adsorb ATPase-FLAG protein, and identifying whether the ATPase-FLAG protein can bind and pull down HNH-HA protein, so as to judge the interaction of the ATPase and HNH protein under physiological conditions.

The SDS-PAGE detection of FIG. 3-A shows that: a theoretical size protein band containing ATPase-FLAG and HNH-HA in the eluted sample of FLAG immunomagnetic beads combined with FIG. 3-B precipitated the FLAG tag protein in both the cleavage supernatant and the Elutation sample, indicating that ATPase-FLAG protein was present in the sample on an objective and efficient precipitation was possible. Subsequent HA precipitation by cleavage of the HNH-HA protein theoretical position on the same membrane revealed that FLAG immunomagnetic bead elution samples contained both ATPase and HNH-HA, which represented that ATPase and HNH had physiological interactions. However, the HNH-HA band was not exposed to the original lysate, and the original HNH content was not high, and the affinity of ATPase was similar to that of the enrichment process. In general, the results of the experiment are consistent with those of the bacterial toxicity experiment and the co-expression experiment, and the fact that ATPase has binding capacity with HNH provides a basis for further optimizing the expression and purification method of HNH protein.

Example 3 optimization of expression purification scheme of HNH protein

Two co-expression plasmids of pCDFDuet-6 XHis-ATPase-HNH and pCDFDuet-ATPase-HNH-6 XHis were constructed first, and the affinity tag was carried on ATPase protein and HNH protein respectively, and the interaction between each other was double verified. The specific steps of protein expression are as follows:

1) Constructing a plasmid. Firstly, constructing two coexpression plasmids of pCDFDuet-6 XHis-ATPase-HNH and pCDFDuet-ATPase-HNH-6 XHis, wherein specific sequences of the 6 XHis-ATPase-HNH and the ATPase-HNH-6 XHis are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively. The sequence of interest was inserted between BamHI/HindIII cleavage sites following the first T7 promoter of the pCDFDuet vector.

2) Plasmid transformation. About 100ng of plasmid is transferred into escherichia coli BL21 (DE 3) expression competent cells, the cells are rapidly placed on ice, the cells are subjected to heat shock at 42 ℃ for 60-90s after being subjected to ice bath for 30min, the cells are placed on the ice for 2-3min, 400 mu L of antibiotic-free LB is added into the cells, and the cells are placed in a shaking table after being uniformly mixed, and are subjected to resuscitative culture for 1h at 37 ℃ and 200 rpm. The culture was centrifuged at 3000rpm for 5min, and after discarding 350. Mu.L of LB, the cell pellet was homogenized with the remaining medium and spread evenly on agar plates of the corresponding plasmid resistance (streptomycin 50. Mu.g/mL), the plates were inverted and incubated overnight at 37 ℃.

3) Culturing in small quantity. The project is selected to be monoclonal in LB with corresponding resistance (50 mug/mL of streptomycin), cultured in a shaking table at 37 ℃ and 220rpm until the OD ₆₀₀ value of the escherichia coli reaches between 0.6 and 0.8, and then added with IPTG with the final concentration of 0.2mM and induced to express for 12 to 16 hours at 18 ℃.

As shown in fig. 4, the difference is that the affinity tag is attached to ATPase and HNH proteins, respectively, to cross-verify the interaction between the two and compare the enrichment effect. The two plasmids are transformed into expression competent cell BL21 (DE 3), and then the culture of the escherichia coli and the expression of the protein are carried out. And carrying out nickel affinity chromatography SDS-PAGE electrophoresis detection on the expressed protein.

The experimental results are shown in FIG. 5-A, when His ₆ tag is carried at the C-terminus of HNH protein, a small amount of expressed HNH protein binds to a corresponding small amount of ATPase protein. When the His ₆ tag is placed on the N-terminus of the ATPase protein, the high-expressed ATPase protein can bring out the low-expressed HNH protein (FIG. 5-B). The expression results of the two expression plasmid proteins show that the HNH proteins pulled down by the tag bands on the ATPase proteins are more (about 2-3mg HNH can be obtained by 6L His ₆ -ATPase bacteria, about 1-2mg HNH can be obtained by 6L His ₆ -HNH bacteria), so that the pCDFDuet-His ₆ -ATPase-HNH plasmid is selected for the expression of HNH proteins subsequently.

Subsequently, we further purified the Ni-NTA initially pure product using Superdex ^TM gel filtration chromatography. As expected, a homogeneous stable complex of ATPase and HNH can be obtained from the initially pure product by gel filtration chromatography, the molecular weight of the ATPase protein is about 62.8kDa, the molecular weight of the HNH protein is about 30.2kDa, and the theoretical peak position is between 11 and 12 mL. As a result, as shown in FIG. 5-C, "Peak1" showed Peak positions between 11 and 12mL, where the theoretical molecular weight of the Peak protein was about 400kDa, and the result of SDS-PAGE was combined to confirm that "Peak1" had and only ATPase protein, and thus "Peak1" was presumed to be hexameric or heptameric ATPase. "Peak 2" is the predominant, most symmetrical protein, with a Peak position of 13-14mL where the theoretical molecular weight of the Peak protein is about 158kDa, and SDS-PAGE results indicate that "Peak 2" has and only ATPase protein, and therefore "Peak 2" is the dimeric ATPase. Peak 3 has a Peak position between 16 and 17mL, where the theoretical molecular weight of the Peak protein is about 29kDa, and SDS-PAGE results show that Peak 3 is mainly monomeric HNH protein.

The co-expression experimental result shows that ATPase and HNH are directly combined, but the combination is unstable or the combination force is not strong, so that the ATPase and HNH can be separated in the molecular sieve analysis process. It is presumed that ATP is not stable, and ATP is continuously consumed during protein purification, and ATPase is subjected to configuration change by ATP hydrolysis, thereby being separated from HNH, and finally, separate HNH protein is obtained. Through the expression purification method, the single HNH protein is successfully obtained, and a foundation is laid for the subsequent HNH enzyme activity research.

EXAMPLE 4 enzyme Activity detection of purified HNH

The genome of the bacterial host and the phage genome are supercoiled, so we have pUC19 plasmid in supercoiled structure as cleavage substrate to examine whether HNH (purified protein) has endonuclease activity and cleave single-or double-stranded DNA. Three groups were set up: supercoiled plasmid (extracted pUC 19), linear plasmid (pUC 19 plasmid treated with 50U commercial EcoRI high fidelity enzyme, linear state), nicked plasmid (pUC 19 treated with 50U commercial BspQI enzyme, nicked state cut into single-stranded gap), correspond to three cases of HNH endo-free enzyme activity, HNH cleavage of double-stranded DNA, HNH cleavage of single-stranded DNA, respectively. Nicked eventually release to an Open spiral state (Open spiral).

As shown in FIG. 6-A, in agarose gel electrophoresis, pUC19 in supercoiled state had the highest electrophoresis rate due to the compact structure, the linearized pUC19 band was positioned in the middle, and pUC19 in open state with loose structure had the lowest electrophoresis rate, and the band was positioned at the top. HNH can cleave 200ng supercoiled pUC19 into Nicked at a protein concentration of 100nM, and 200ng pUC19 can be cleaved almost completely at a concentration of 1. Mu.M, with cleavage products in the Linear state, but mainly in the Nicked state, indicating that HNH has a nicking enzyme activity for cleavage of single-stranded DNA (NICKASE ACTIVITY).

The activity of HNH endonucleases is known to have the characteristic of being dependent on divalent metal ions Mg ²⁺, so that the influence of some common divalent metal ions on the nicking enzyme activity of HNH is tested next, the control group is untreated pUC19 and a reaction system added with a metal ion chelating agent EDTA, and the experimental group contains 1mM metal ions, so that as shown in FIG. 6-B, the cleavage activity of HNH can be promoted by Mg ²⁺、Mn² ⁺、Ca²⁺、Ni²⁺ except Zn ²⁺.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the embodiments described above will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended 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. An HNH toxic protein expression vector comprising an ATPase coding box and an HNH nuclease coding box, wherein the ATPase coding box and HNH nuclease coding box are simultaneously independently expressible.

2. The vector of claim 1, wherein the ATPase coding box and HNH coding box nucleases are both ATPase coding box and HNH nuclease coding box from e.coli Retron-Eco4 system.

3. The vector of claim 1, wherein the ATPase coding box is linked to a tag protein coding box, which together with the ATPase coding box expresses a fusion protein comprising the tag protein and the ATPase.

4. The vector of claim 1, wherein the HNH coding box nuclease is downstream of the ATPase coding box and comprises a spacer sequence therebetween, said spacer sequence allowing independent expression of the HNH nuclease coding box and the ATPase coding box.

5. A method for purifying HNH toxic protein expression, the method comprising the steps of:

1) The HNH nuclease coding frame and the ATPase coding frame are simultaneously and independently expressed in the same cell.

2) And (5) primarily purifying the expression product.

3) The initially purified product was further purified.

6. The method according to claim 5, wherein the HNH nuclease coding box and the ATPase coding box in step 1) are simultaneously and independently expressed in the same vector.

7. The method according to claim 5, wherein the ATPase coding box and the HNH coding box in step 1) are ATPase nuclease coding box and HNH coding box from the E.coli Retron-Eco4 system.

8. The method according to claim 5, wherein the ATPase coding box in step 1) is linked to a tag protein coding box, and the tag protein coding box and the ATPase coding box together express a fusion protein comprising the tag protein and the ATPase.

9. An endonuclease, the sequence of which comprises an amino acid sequence as shown in the sequence SEQ ID NO. 1.

10. Use of a protein comprising the amino acid sequence shown in SEQ ID No.1 for the preparation of an endonucleolytic enzyme.