CN118240856A - Escherichia coli toxic protein and its use and prokaryotic expression method - Google Patents

Abstract

The present invention provides a highly efficient expression and purification method of a cytotoxic protein HNH having endonuclease activity, and proves 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

Escherichia coli toxic protein and its use and prokaryotic expression method

Application technology

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

Background technique

HNH enzymes are also called "ββα-me" endonucleases. All members of this family have a highly similar catalytic motif - a ββα topological structure, which consists of 30 to 40 amino acids and is responsible for the binding and cleavage of nucleic acids. The structural feature is that three conserved amino acid His-Asn-His residues (H-N-H) fold into a β-α-metal topological structure with two antiparallel β sheets, an α helix and a divalent metal cation binding, and finally folds into a ββα-metal topological structure.

The sequence conservation of HNH proteins is reflected in the conserved His at the end of the β1 chain (corresponding to the first letter "H" in HNH), the metal ion binding to the highly conserved Asn/His residue located in the α-helix (corresponding to the last letter "H" in HNH), the conserved Asn at the beginning of the β2 chain (corresponding to the letter "N" in HNH), and the highly conserved Asn usually forms hydrogen bonds with the main chain atoms next to the catalytic His, thus playing an important 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 foreign or host genomes (Nickase endonucleases), and when they function as homodimers, they introduce double-stranded DNA breaks for DNA restriction, integration, recombination, and repair.

Previous studies have shown that when the HNH protein is expressed in prokaryotes, the experimental phenomenon is that E. coli can be cultured, but once induced to express, the bacterial solution gradually becomes clear, and it is preliminarily speculated that the HNH protein is cytotoxic. Therefore, establishing an expression and purification system that does not produce cytotoxicity is the key to improving the expression efficiency of the HNH protein. In addition, the HNH protein family has many different sequences, and the identification of new HNH proteins with endonuclease activity can further enrich the number of tool enzymes and provide more options for the modification and optimization of tool enzymes.

Summary of the invention

In view of the problems existing in the prior art, the embodiments of the present invention provide a method for co-expressing an HNH protein with an ATPase protein in the same open reading frame to obtain a soluble target HNH protein.

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

Furthermore, the HNH toxic protein expression vector is composed of an ATPase coding frame and a HNH coding frame inserted into any prokaryotic expression vector.

Furthermore, the HNH toxic protein expression vector is composed of an ATPase coding frame and an HNH coding frame inserted into a position in any prokaryotic expression vector where the ATPase coding frame and the HNH coding frame can be expressed.

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

Furthermore, the ATPase coding frame and the HNH coding frame are both from the ATPase coding frame and the HNH coding frame of the Retron-Eco4 system of the same bacterium.

Furthermore, the ATPase coding frame and the HNH coding frame are both from the ATPase coding frame and the HNH coding frame of the Retron-Eco4 system of the same bacterial strain.

Furthermore, the protein encoded by the ATPase coding frame and the protein encoded by the HNH coding frame interact with each other, and the protein encoded by the ATPase coding frame can inhibit the cytotoxicity of the protein encoded by the HNH coding frame.

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 be separated during the molecular sieve chromatography process.

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

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

In a preferred embodiment, the HNH coding frame is located downstream of the ATPase coding frame, and a spacer sequence is contained between the two, and the spacer sequence allows the HNH coding frame and the ATPase coding frame to be expressed independently.

In a preferred embodiment, the HNH coding frame, ATPase coding frame, spacer sequence and tag protein coding frame form the following structure: F-ATPase-B-HNH or ATPase-B-HNH-F, wherein 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).

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.

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

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

A method for expressing and purifying a HNH toxic protein, the method comprising the following steps:

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

2) Preliminary purification of the expression product.

3) The preliminary purified product is further purified.

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

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

Furthermore, the ATPase coding frame and the HNH coding frame in step 1) are both ATPase coding frame and HNH coding frame from the Retron-Eco4 system of the same bacterium.

Furthermore, the ATPase coding frame and the HNH coding frame in step 1) are both ATPase coding frame and HNH coding frame from the Retron-Eco4 system of the same bacterial strain.

Furthermore, in step 1), the protein encoded by the ATPase coding frame and the protein encoded by the HNH coding frame interact with each other, and the protein encoded by the ATPase coding frame can inhibit the cytotoxicity of the protein encoded by the HNH coding frame.

Furthermore, in step 1), 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 be separated during the molecular sieve chromatography process.

Furthermore, in step 1), the ATPase coding frame or the HNH coding frame is connected to a tag protein coding frame, and the tag protein coding frame and the ATPase coding frame or the HNH coding frame together express a fusion protein containing the tag protein and ATPase or HNH, and the tag protein is located at the 5' or 3' end of the ATPase coding frame.

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

In a preferred embodiment, the HNH coding frame is located downstream of the ATPase coding frame, and a spacer sequence is contained between the two, and the spacer sequence allows the HNH coding frame and the ATPase coding frame to be expressed independently.

In a preferred embodiment, the HNH coding frame, ATPase coding frame, spacer sequence and tag protein coding frame form the following structure: F-ATPase-B-HNH or ATPase-B-HNH-F, wherein 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).

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.

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

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

In a preferred embodiment, step 3) is purified by gel filtration chromatography.

A restriction endonuclease, the sequence of which contains the amino acid sequence shown in SEQ ID NO.1.

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

Furthermore, the endonuclease has nickase activity for cutting single-stranded DNA.

The present invention has the following beneficial effects:

1. A new vector and purification method for the expression and purification of toxic proteins are provided, which improves the expression efficiency of toxic proteins.

2. Provides a new enzyme with nickase activity for cutting single-stranded DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific embodiments.

Figure 1: Construction of expression plasmids required for cytotoxicity experiments.

Figure 2: Cytotoxicity test results. The bacterial concentration values decrease from top to bottom, and the images shown are representative images of three repeated experiments. (A) HNH is cytotoxic; (B) ATPase inhibits HNH cytotoxicity.

Figure 3: Co-IP experimental results 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.

Fig. 4. Construction of ATPase and HNH co-expression plasmid.

Figure 5: Results of co-expression of ATPase and HNH. (A) Nickel affinity chromatography SDS-PAGE results of co-expression of the tag band on HNH; (B) Nickel affinity chromatography SDS-PAGE results of co-expression of the tag band on ATPase; (C) Superdex200 gel filtration chromatography purification results of the co-expression product of ATPase and HNH.

Figure 6: HNH endonuclease activity results. (A) Nickase activity in the presence of 100 nM HNH protein; (B) Effects of different divalent cations on HNH activity; (C) Effects of different nucleotides on HNH activity; (D) Schematic diagram of endonuclease assay.

Figure 7: Changes in bacterial solution after adding IPTG to induce expression. 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.

Detailed ways

The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work 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 those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention in this specification are only for the purpose of describing specific embodiments and are not used to limit the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified.

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.

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 1 HNH cytotoxicity experiment and its cytotoxicity inhibition mechanism

During the protein expression process, we inserted the target gene of HNH protein (SEQ ID NO.1) into the BamHI/HindIII restriction sites of pET28 vector (SEQ ID NO.6) to construct a plasmid expressing HNH protein, and transferred the plasmid into Escherichia coli BL21 (DE3) bacteria for expression. It was found that normal plaques could grow on the plate, and the OD ₆₀₀ value of the bacteria could be normally cultured to 0.6-0.8 when a single clone was selected for culture. However, once IPTG was added to induce expression, the bacterial solution would gradually change from turbid to clear. As shown in the figure: the bacteria in the left centrifuge tube became clear after 1 hour of induction with 0.2mM IPTG, while the uninduced bacteria on the right were still turbid after 1 hour (see Figure 7).

The Retron system functions as a whole in E. coli, and the HNH protein may be cytotoxic, so we speculated that under normal physiological conditions, if the Retron-Eco4 complete system is not toxic, then there is a component in the system that inhibits the toxicity of HNH. To verify this speculation, we performed single-component deletion mutation and bacterial toxicity experiments.

In order to identify the components that inhibit the toxicity of HNH in the Retron-Eco4 system, different primers were designed (as shown in Table 1), and the Retron-Eco4 complete component plasmid and single-component deletion plasmid (ΔRT, ΔATPase, ΔHNH) were constructed. As shown in Figure 1, all genes were cloned into the pCDFDuet vector and then uniformly transformed into BL21 (DE3) bacteria for cytotoxicity testing.

Given that the effector protein ADP-ribosyltransferase in the Retron-Sen2 system is a toxin protein with low temperature toxicity, the effector protein ADP-ribosyltransferase of Retron-Eco9 is cytotoxic under both 37°C and low temperature conditions (below 25°C). Therefore, this study explored whether HNH is toxic at different temperatures of 37°C and 18°C. In addition, because the inducer in the experiment is IPTG, IPTG itself has a certain toxicity to the growth of bacteria. In order to exclude this effect, this experiment referred to the experimental method of the Retron-Sen2 system in Nature magazine in 2022 (Jacob Bobonis et al, Bacterial retrons encodephage-defending tripartite toxin–antitoxin systems, 2022, Nature), and set up an uninduced group (no IPTG), a low concentration IPTG group (0.1mM), and a high concentration IPTG group (1mM). Three groups of parallel replicates were set for each experimental condition.

Table 1: Systematic single-component deletion mutation primers

The experimental results in Figure 2-A show that: (1) the number of plaques of E. coli containing HNH plasmid in the low-concentration IPTG group and the high-concentration IPTG group was significantly reduced compared with the uninduced group, and the morphology was also uneven; (2) bacteria containing the Retron-Eco4 complete system plasmid grew well in the uninduced group and the low-concentration IPTG group, but did not grow well in the high-IPTG group due to the toxicity of IPTG; (3) However, it can 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 Retron-Eco4, which can rule out the toxic effect of high IPTG concentration on bacteria. Therefore, the experimental results show that HNH is cytotoxic under different temperature conditions, while the complete Retron-Eco4 system is not cytotoxic under different temperature conditions.

In Figure 2-B, after low IPTG induction (IPTG low), the number of plaques of bacteria containing ΔRT and ΔHNH plasmids under different temperature conditions showed no obvious cytotoxicity compared with the plaques of the complete Retron-Eco4 system (IPTG low) in Figure 2-A, and was greater than the number of plaques of HNH alone in Figure 2-A (IPTG low), which was consistent with the high IPTG group; however, bacteria containing ΔATPase plasmids showed different plaque sizes under different temperature conditions, and the plaques after high IPTG induction and low IPTG induction were significantly reduced compared with the complete Retron-Eco4 system. Therefore, it was concluded that in the complete system, when ATPase was missing, the toxic effect of HNH was released, which can be understood as ATPase inhibiting the toxicity of HNH.

In summary, the results of the cytotoxicity experiment showed that the complete Retron-Eco4 system was not toxic, HNH expressed alone was cytotoxic at different temperatures, and ATPase showed HNH toxicity inhibition at different temperatures, indicating that ATPase and HNH may interact under physiological conditions, and further suggesting that co-expression of ATPase and HNH is an important way to obtain soluble HNH protein.

Example 2 HNH interacts with ATPase

This project first constructed the pCDFDuet-ATPase-FLAG-HNH-HA expression plasmid, as shown in Figure 3-A, with a FLAG tag at the C-terminus of ATPase and an HA tag at the C-terminus of HA protein. The constructed plasmid was then transformed into E. coli BL21 (DE3) expression competent cells, 0.2mM IPTG induced expression of 40-50mL bacterial solution, and the cells were collected and ultrasonically disrupted on ice and then centrifuged at low temperature to obtain the supernatant containing soluble ATPase-FLAG and HNH-HA proteins. Subsequently, the ATPase-FLAG protein was adsorbed using FLAG-tagged immunomagnetic beads, and then it was determined whether the ATPase-FLAG protein could bind to and pull down the HNH-HA protein, so as to determine the interaction between ATPase and HNH protein under physiological conditions.

The SDS-PAGE test results in Figure 3-A show that the eluted sample of FLAG immunomagnetic beads contains protein bands of theoretical sizes of ATPase-FLAG and HNH-HA. Combined with the precipitated FLAG-tagged protein in both the lysate supernatant and the elution sample in Figure 3-B, it indicates that the ATPase-FLAG protein objectively exists in the sample and can be effectively precipitated. The theoretical position of the HNH-HA protein on the same membrane was cut for subsequent HA precipitation. The results showed that the eluted sample of FLAG immunomagnetic beads contained both ATPase and HNH-HA, which means that ATPase and HNH have physiological interactions. However, no HNH-HA band was exposed in the original lysate. It is speculated that the original HNH content is low, and the affinity of ATPase is similar to an enrichment process. In general, this experiment is consistent with the results of the aforementioned bacterial toxicity experiment and co-expression experiment, proving that ATPase and HNH have the ability to bind, which provides a basis for further optimization of the expression and purification method of HNH protein.

Example 3 Optimization of the expression and purification method of HNH protein

First, construct two co-expression plasmids, pCDFDuet-6×His-ATPase-HNH and pCDFDuet-ATPase-HNH-6×His. This step is to carry affinity tags on the ATPase protein and HNH protein respectively to double verify the interaction between them. The specific steps of protein expression are as follows:

1) Plasmid construction. First, construct two co-expression plasmids, pCDFDuet-6×His-ATPase-HNH and pCDFDuet-ATPase-HNH-6×His. 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 is inserted between the BamHI/HindIII restriction sites after the first T7 promoter of the pCDFDuet vector.

2) Plasmid transformation. In the experiment, about 100 ng of plasmid was transferred into the expression competent cells of E. coli BL21 (DE3), and the cells were quickly placed on ice. After ice bathing for 30 minutes, they were heat-shocked at 42°C for 60-90 seconds and placed on ice for 2-3 minutes. Then 400 μL of LB without antibiotics was added to the cells, mixed and placed in a shaker, and revived and cultured at 37°C and 200 rpm for 1 hour. The culture was centrifuged at 3000 rpm for 5 minutes, and 350 μL of LB was discarded. The cell pellet was mixed with the remaining culture medium and evenly spread on the agar plate corresponding to the plasmid resistance (streptomycin 50 μg/mL). The plate was inverted and cultured in an incubator at 37°C overnight.

3) Small-scale culture. In this project, a single clone was selected in LB with corresponding resistance (streptomycin 50 μg/mL), and cultured in a shaker at 37°C and 220 rpm until the OD ₆₀₀ value of E. coli reached between 0.6 and 0.8, and then IPTG was added at a final concentration of 0.2 mM and induced for expression at 18°C for 12-16 hours.

As shown in Figure 4, the difference is that the affinity tags are respectively carried on the ATPase and HNH proteins to cross-validate the interaction between the two and compare the enrichment effect. The two constructed plasmids were transformed into expression competent cells BL21 (DE3), and then Escherichia coli was cultured and the protein was expressed. The expressed protein was detected by nickel affinity chromatography SDS-PAGE electrophoresis.

The experimental results are shown in Figure 5-A. When the His ₆ tag is at the C-terminus of the HNH protein, a small amount of HNH protein expressed binds to a corresponding small amount of ATPase protein. When the His ₆ tag is at the N-terminus of the ATPase protein, a large amount of ATPase protein expressed can bring out a small amount of HNH protein expressed (Figure 5-B). The protein expression results of the two expression plasmids showed that the tag pulled down more HNH protein on the ATPase protein (6L His ₆ -ATPase bacteria can obtain about 2-3mg HNH, 6L His ₆ -HNH bacteria can obtain about 1-2mg HNH), so the pCDFDuet-His ₆ -ATPase-HNH plasmid was selected for the expression of HNH protein.

Subsequently, we further purified the Ni-NTA primary pure product using Superdex ^TM 200 gel filtration chromatography. As expected, the primary pure product can obtain a uniform and stable complex of ATPase and HNH through gel filtration chromatography. The molecular weight of ATPase protein is about 62.8kDa, and the molecular weight of HNH protein is about 30.2kDa. The theoretical peak position is between 11-12mL. The results are shown in Figure 5-C. The peak position of "Peak 1" is between 11-12mL. The theoretical molecular weight of the peak protein here is about 400kDa. Combined with the SDS-PAGE results, it is confirmed that "Peak 1" has and only has ATPase protein, so it is speculated that "Peak 1" is a hexameric or heptameric ATPase. "Peak 2" is the most important and symmetrical protein, with a peak position of 13-14mL. The theoretical molecular weight of the peak protein here is about 158kDa. The SDS-PAGE results show that "Peak 2" has and only has ATPase protein, so "peak2" is a dimeric ATPase. The peak position of "Peak 3" is between 16-17mL. The theoretical molecular weight of the peak protein here is about 29kDa. The SDS-PAGE results show that "Peak 3" is mainly monomeric HNH protein.

The results of the co-expression experiment showed that ATPase and HNH bind directly, but the binding is unstable or weak, so they are separated during the molecular sieve chromatography process. It is speculated that due to the unstable nature of ATP, ATP is continuously consumed during the protein purification process, and the conformation of ATPase changes due to ATP hydrolysis, thereby separating from HNH and finally obtaining a single HNH protein. Through this expression and purification method, we successfully obtained a single HNH protein, laying the foundation for 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 structures, so we used the supercoiled pUC19 plasmid as a cutting substrate to detect whether HNH (purified protein) has endonuclease activity and whether it cuts single-stranded or double-stranded DNA. The experiment set up three groups: supercoiled plasmid (extracted pUC19), linear plasmid (pUC19 plasmid treated with 50U commercial EcoRI high-fidelity enzyme, Linear state), Nicked plasmid (pUC19 treated with 50U commercial BspQI enzyme, Nicked state with single-stranded gaps), corresponding to three situations: HNH has no endonuclease activity, HNH cuts double-stranded DNA, and HNH cuts single-stranded DNA. Nicked is finally released into an open spiral state (Open circular).

The experimental results are shown in Figure 6-A. In agarose gel electrophoresis, the supercoiled pUC19 has the fastest electrophoresis rate due to its compact structure. The linearized pUC19 band is in the middle. The loosely structured open pUC19 has the slowest electrophoresis rate, with the band at the top. HNH can cut 200ng of supercoiled pUC19 into a Nicked state at a protein concentration of 100nM, and can basically cut 200ng of pUC19 completely when the concentration reaches 1μM. There are cleavage products in the Linear state, but mainly in the Nicked state, indicating that HNH has nickase activity for cutting single-stranded DNA.

It is known that the activity of HNH endonuclease is dependent on the divalent metal ion ^Mg2+ , so the effects of some common divalent metal ions on the incision activity of HNH were tested next. The control group was untreated pUC19 and the reaction system with the addition of metal ion chelator EDTA. The experimental group contained 1 mM metal ions. The results are shown in Figure 6-B. Except for Zn2 ⁺ , ^Mg2+ , ^{Mn2 +} , Ca2 ⁺ , and Ni2 ⁺ can all promote the cutting activity of HNH.

The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. An HNH toxic protein expression vector, the vector comprising an ATPase coding frame and an HNH nuclease coding frame, and the ATPase coding frame and the HNH nuclease coding frame can be expressed independently at the same time. 2. The vector according to claim 1, characterized in that the ATPase coding frame and the HNH coding frame nuclease are both ATPase coding frame and HNH nuclease coding frame from the Escherichia coli Retron-Eco4 system. 3. The vector according to claim 1 is characterized in that the ATPase coding frame is connected to a tag protein coding frame, and the tag protein coding frame and the ATPase coding frame together express a fusion protein containing the tag protein and ATPase. 4. The vector according to claim 1 is characterized in that the HNH nuclease coding frame is located downstream of the ATPase coding frame, and a spacer sequence is contained between the two, and the spacer sequence can make the HNH nuclease coding frame and the ATPase coding frame express independently. 5. A method for expressing and purifying a HNH toxic protein, the method comprising the following steps: 1) The HNH nuclease coding frame and ATPase coding frame are expressed independently and simultaneously in the same cell. 2) Preliminary purification of the expression product. 3) The preliminary purified product is further purified. 6. The method for expressing and purifying HNH toxic protein according to claim 5, characterized in that in step 1), the HNH nuclease coding frame and the ATPase coding frame are independently expressed simultaneously in the same vector. 7. The method for expressing and purifying HNH toxic protein according to claim 5, characterized in that the ATPase coding frame and the HNH coding frame in step 1) are both ATPase nuclease coding frame and HNH coding frame from the Escherichia coli Retron-Eco4 system. 8. The method for expressing and purifying HNH toxic protein according to claim 5, characterized in that the ATPase coding frame in step 1) is connected to a tag protein coding frame, and the tag protein coding frame and the ATPase coding frame together express a fusion protein containing the tag protein and ATPase. 9. An endonuclease, the sequence of which contains the amino acid sequence shown in SEQ ID NO.1. 10. Use of a protein comprising the amino acid sequence shown in SEQ ID NO. 1 in the preparation of an endonuclease.