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Adenosine monophosphate enzyme as well as preparation method and application thereof

A technology of adenylyl monophosphate and adenosine monophosphate, which is applied in the fields of molecular biology and biochemistry, can solve the problems of unclear functions of non-exocrine Fic protein, and achieve mass production and simple preparation methods , The effect of low purification cost

Pending Publication Date: 2022-02-15
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although the catalytic mechanism of the exocrine Fic protein of a few pathogenic bacteria has been clarified, the function of the bacterial non-exocrine Fic protein is still unclear

Method used

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  • Adenosine monophosphate enzyme as well as preparation method and application thereof
  • Adenosine monophosphate enzyme as well as preparation method and application thereof
  • Adenosine monophosphate enzyme as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The core of Example 1 is to provide the cloning of adenylyl monophosphate enzyme gene.

[0039] Such as figure 1As shown, the cloning of the adenylyl monophosphate gene mainly includes the steps of bacterial activation and cultivation, DNA extraction, PCR amplification and detection, enzyme digestion, ligation, transformation and sequencing analysis. The specific details are as follows:

[0040] S1: Bacterial activation and cultivation

[0041] Pick a little biocontrol Pseudomonas fluorescens (P.fluorescens) 2P24 from the -80°C refrigerator with a sterile tip, and streak it on the LA plate (each liter of medium contains 5g NaCl, 10g tryptone, 5g yeast powder, 16g agar powder, pH = 7.2), placed at 28°C and cultured in the dark for 48 hours, picked a single colony in 2.5mL of LB medium (each liter of medium contained 5g NaCl, 10g tryptone, 5g yeast powder, pH = 7.2) in a sterile centrifuge tube, placed in an air-bath shaker at 28°C and 180r / min for 24h.

[0042] S2: D...

Embodiment 2

[0053] The core of Example 2 is to provide expression and purification of adenylyl monophosphate enzyme.

[0054] Adenylyl monophosphate must be induced and expressed in a small amount to verify whether the protein can be expressed, the expression and its size analysis, and then induce expression in a large amount after confirmation. The expression and purification of adenylyl monophosphate enzyme are detailed as follows:

[0055] Step 1: Protein Expression

[0056] The expression vector pET-22b(+)::fic-1 was transformed into chemically competent cells BL21(DE3) by heat shock, and placed in a 37°C constant temperature incubator for 20 hours after plating, and a single colony was inoculated and shaken to induce a small amount to confirm protein expression Then pick a single colony and inoculate it into 12.5mL liquid LB medium containing 100μg / mL ampicillin, shake culture at 37°C for 6-8h, and transfer to a 2L Erlenmeyer flask containing 500mL liquid LB medium (containing 100μg...

Embodiment 3

[0068] The core of this embodiment 3 is to provide adenosine monophosphate modified Escherichia coli GyrB, GyrB 1-200 . In this embodiment 3, except that the following experiments and results are different, all the other operating steps are the same as in embodiments 1 and 2.

[0069] 1. Fic-1 H135A The mutant construction is based on the pET-22b(+)::fic-1 vector, and the single-base mutation technique is used to mutate the 135th histidine of the Fic-1 protein to alanine to obtain pET-22b(+):: fic-1 H135A ;

[0070] 2. Cloning E. coli EcGyrB, N-terminal ATPase domain EcGyrB 1-200 , C-terminal Tranducter and Toprim domain EcGyrB 201-804 , using the genome of Escherichia coli MG1655 strain as a template;

[0071] 3. Before adding IPTG to induce protein expression, the cell OD 600 It is 0.6-0.8.

[0072] 4. Monophosphate adenylation reaction: Prepare reaction system on ice, 20 μL reaction system contains 1.5 μg Fic-1 protein, 10 μg EcGyrB or EcGyrB 1-200 or EcGyrB 201-8...

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Abstract

The invention discloses adenosine monophosphate enzyme as well as a preparation method and an application thereof, including an expression vector of adenosine monophosphate enzyme, a preparation method of the adenosine monophosphate enzyme, an application of the adenosine monophosphate enzyme in in-vitro adenosine monophosphate modification of subunit GyrB and ParE proteins of DNA topoisomerase, and an application of the adenosine monophosphate enzyme in in-vitro inhibition of ATP hydrolysis activity of the DNA topoisomerase, and in inhibition of DNA topoisomerase negative superhelix and relaxation of DNA activity. The preparation method of the adenosine monophosphate enzyme provided by the invention is simple, short in fermentation period, low in purification cost and large in preparation amount; by utilizing the adenosine monophosphate enzyme provided by the invention, adenosine monophosphate modification of the DNA topoisomerase can be realized under an in-vitro condition; the adenosine monophosphate enzyme provided by the invention is expressed, the activity of DNA topoisomerase can be inhibited.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and biochemistry, in particular to an adenylyl monophosphate enzyme and its preparation method and application. Background technique [0002] Protein post-translational modification refers to the loading of specific groups or the removal of certain amino acids on protein amino acid residues. Post-translational modification of proteins can either add different groups (such as phosphoric acid, acetyl, methyl or polysaccharides, etc.) or signal peptide) cleavage. Modification can change protein function, structure, stability, positioning or affect the interaction between protein and other molecules, thereby regulating cell metabolism and physiology; the modified group can be a small molecule or a small protein; there are reversible modifications, There are also irreversible modifications; the result of the modification may or may not be beneficial for bacterial growth. Post-translationa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/70C12N15/55C12N1/21C12R1/19
CPCC12N9/14C12N15/70
Inventor 卢灿华夏振远马俊红盖晓彤姜宁晋艳
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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