Nucleoside triphosphate hydrolase as well as purification method and application thereof

A technology of nucleoside triphosphate water and nucleoside phosphate water, which is applied in the field of molecular biology, can solve the problems of poor turnover rate, unrevealed GajB function, insufficient resistance to phage invasion, etc., and achieve the effect of improving purity

Pending Publication Date: 2022-06-21
HUAZHONG UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, the function of GajA protein has been analyzed (Chengand Zhu et al., A nucleotide-sensing endonuclease from the Gabija bacterial defense system. Nucleic acids research, 2021, 49(9)), but its activity is inhibited by nucleotides and its The turnover rate is r...

Method used

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  • Nucleoside triphosphate hydrolase as well as purification method and application thereof
  • Nucleoside triphosphate hydrolase as well as purification method and application thereof
  • Nucleoside triphosphate hydrolase as well as purification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Interaction research between GajB protein and GajA protein, discovery and functional research of GajB' protein

[0053] 1. Detection of the interaction between GajB protein and GajA protein

[0054] (1) The coding gene of GajA protein and the coding gene of GajB protein are driven by a promoter to induce expression (the target protein is denoted as GajAB protein)

[0055]Gene recombination: the Gabija bacterial defense system includes GajA protein and GajB protein (as shown in SEQ ID No.1) two components, using the method of gene synthesis (gene synthesis is completed by Nanjing GenScript Biotechnology Co., Ltd.) to obtain the Gabija system The coding sequence (as shown in SEQ ID No.6), it is cloned between the restriction sites Nde I and Not I of the prokaryotic expression vector pET-28a, and its gene 5' end is fused with a 60-base length DNA fragment, transform the obtained recombinant vector into E.coli BL21 (DE3) cells to obtain competent cells transform...

Embodiment 2

[0067] Example 2: GajB and GajB' protein coding gene amplification, nucleoside triphosphate hydrolase purification method

[0068] Gene amplification and protein expression of S1, GajB and GajB' proteins

[0069] GajB gene and GajB' gene were obtained by gene synthesis (gene synthesis was completed by Nanjing KingScript Biotechnology Co., Ltd.), and GajB gene (or GajB' gene) was cloned into the restriction site of prokaryotic expression vector pET-28a Between Nde I and Not I, the 5' end of its gene is fused with a DNA fragment of 60 base lengths, which encodes GajB protein (or GajB' protein), histidine tag (as shown in SEQ ID No.5 ), and the flexible peptide between the histidine tag and the GajB protein (or GajB' protein) (as shown in SEQ ID No.9), the resulting recombinant vector was transformed into E.coli BL21 (DE3) cells, Competent cells transformed with recombinant plasmids were obtained.

[0070] At 37°C, place the competent cells transformed with the recombinant plas...

Embodiment 3

[0077] Example 3: In vitro function of purified nucleoside triphosphate hydrolase

[0078] 1. Detection of activity of nucleoside triphosphate hydrolase after purification in Example 2

[0079] In the following experiments, PiColorLock was used to detect the hydrolysis activity of GajB' protein or GajB protein on nucleoside triphosphate TM Phosphate detection kit (purchased from EXPEDEON company), the operation method refers to the kit manual.

[0080] (1) Effect of temperature on the activity of nucleoside triphosphate hydrolase

[0081] In order to explore the activity of the nucleoside triphosphate hydrolase purified in Example 2, the inventors first studied the effect of the reaction temperature on the activity of the nucleoside triphosphate hydrolase. The reaction system was as follows: water, 6.5 μL; 10× reaction buffer, 1 μL; 5mMATP, 1μL; 5μM ssDNA, 1μL; 10μM protein, 0.5μL; the total volume of the reaction system was 10μL; the protein was GajB' protein or GajB prote...

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Abstract

The invention provides nucleoside triphosphate hydrolase as well as a purification method and application thereof, and belongs to the technical field of molecular biology. The nucleoside triphosphate hydrolase is GajB protein or GajB'protein, and the nucleoside triphosphate hydrolase is used for hydrolyzing nucleoside triphosphate in the presence of metal ions and DNA (deoxyribonucleic acid) at the same time; the amino acid sequence of the GajB protein is as shown in SEQ ID No. 1, and the amino acid sequence of the GajB'protein is as shown in SEQ ID No. 2; the metal ions are magnesium ions, manganese ions and calcium ions. The purity of the nucleoside triphosphate hydrolase is improved by connecting the identification tag to the amino terminal of the GajB protein or the GajB'protein and selecting a proper purification method. The nucleoside triphosphate hydrolase can be applied to the fields of DNA detection, DNA processing, gene editing, gene engineering and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a nucleoside triphosphate hydrolase, a purification method and application thereof. Background technique [0002] Nucleoside triphosphate is one of several structures of nucleotides. According to the number of phosphate groups in the nucleotide molecule, the molecular structure of nucleotides is nucleoside monophosphate (NMP), nucleoside diphosphate (NDP) and triphosphate Nucleosides (NTPs). Nucleosides are condensates formed by purine or pyrimidine bases and ribose. The five hydroxyl groups on the ribose of this condensate form lipids with tripolyphosphate to form nucleoside triphosphates. At the same time, they are also nucleotides. synthetic substrate. Nucleoside triphosphate is a nucleotide containing three phosphate groups. The common forms in nature include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymid...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/70
CPCC12N9/14C12N15/70C12Y306/01
Inventor 朱斌成锐
Owner HUAZHONG UNIV OF SCI & TECH
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