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Novel temperature-response inorganic pyrophosphatase conjugate synthesis and application of conjugate to enhanced polymerase chain reaction

An inorganic pyrophosphatase, temperature-responsive technology, used in the determination/inspection of microorganisms, hydrolase, biochemical equipment and methods, etc., can solve the problem of screening and extraction of thermophilic microorganisms denaturation inactivation and other problems, to achieve the effect of improving the optimum reaction temperature and heat resistance, easy control of the process, and simple process method

Active Publication Date: 2015-08-26
SUZHOU UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Although it is possible to extract PPase with high temperature resistance properties from some high temperature resistant thermophilic bacteria in nature, it is very difficult to obtain, screen, and extract such thermophilic microorganisms, and special environmental requirements are required for their cultivation and expression. The heat-resistant enzymes that have been commercialized, such as PPase extracted from Bacillus stearothermophilus, have much lower enzyme activity than PPase usually extracted from Escherichia coli
Escherichia coli is commonly used as an engineering bacterium, and the PPase enzyme activity extracted from it exceeds 800 units / mg, showing its high activity characteristics, but its optimum reaction temperature is only 45-50 o About C, not only can’t meet the requirement of PCR reaction temperature, but also the protein is easily denatured and inactivated under high temperature conditions, which requires modifying the PPase of Escherichia coli to improve its optimum reaction temperature and heat resistance. PCR efficiency has great potential

Method used

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  • Novel temperature-response inorganic pyrophosphatase conjugate synthesis and application of conjugate to enhanced polymerase chain reaction
  • Novel temperature-response inorganic pyrophosphatase conjugate synthesis and application of conjugate to enhanced polymerase chain reaction
  • Novel temperature-response inorganic pyrophosphatase conjugate synthesis and application of conjugate to enhanced polymerase chain reaction

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment one, the result is as figure 1 with 2 shown.

[0041] (1) Expression and extraction of the mutant (K148C): a site-directed mutagenesis method was used to introduce a cysteine ​​containing a sulfhydryl group near the active center of the protein.

[0042] (2) Preparation of polymer PNIPAM: adjust NIPAM and CTA seasoning ratio at 65 o C for 24 h, followed by dialysis and lyophilization to obtain polymers with different molecular weights. Reversible addition-fragmentation chain transfer polymerization (RAFT)

[0043] (3) Preparation of polymers with thiopyridine: add ethanolamine and dithiobipyridine, react at room temperature for 4 h, dialyze and freeze-dry to obtain the modified polymer Pydl-PNIPAM.

[0044] (4) Preparation of PNI-PPase conjugate: Combine in 10 mM Tris-HCl buffer solution (pH=8.0), the molar ratio of PNIPAM / PPase is 50 / 1, and react overnight to obtain the conjugate PNI-PPase.

Embodiment 2

[0045] Embodiment two, the result is as image 3 Shown is the activity test of the conjugate at different temperatures and 60 o Activity test after different incubation times at C.

[0046] (1) Determination of the activity of the conjugate at different temperatures: the conjugate was heated at 25-90 o C for 10 min heat treatment, and then with the substrate sodium pyrophosphate (Na 4 P 2 o 7 ) at the corresponding temperature for 10 min, measure the Pi content of the product, and calculate the specific activity value (kat / kg).

[0047] (2) Determination of the thermal stability of the conjugate at different times: the conjugate was heated at 60 o After heat treatment at C for 10-180 min, the activity was measured, and the specific activity value and relative activity were calculated (the specific activity value at the time of heat treatment for 10 min was 100%).

Embodiment 3

[0048] Embodiment three, the result is as Figure 4 Shown is the enhancement effect of the conjugate added to the PCR system and the activity test after different cycles of heat treatment in the PCR instrument.

[0049] (1) PCR enhancement effect test: PPase, PNIPAM, and PNI-PPase were added to the PCR system, and ordinary PCR was used as a control to measure the amplification after 20 cycles.

[0050] (2) Changes in the activity of the conjugate under different cycles of the PCR instrument: place the conjugate in the PCR instrument for 20-100 cycles, measure the activity of the conjugate, and calculate the specific activity value (kat / kg) , which is sodium pyrophosphate (Na 4 P 2 o 7 ) as the substrate.

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Abstract

The invention relates to novel temperature-response inorganic pyrophosphatase conjugate synthesis and application of the conjugate to enhanced polymerase chain reaction, belongs to the field of polymer-protein coupling techniques and application of the polymer-protein coupling techniques to biology, and provides a strategy of enhancing PCR (polymerase chain reaction) efficiency. By the aid of the polymer-protein coupling techniques, temperature-response PNIPAM (polymer N-isopropylacrylamide) is modified in a site-directed manner near the active center of inorganic PPase (pyrophosphatase), heat stability of protein is enhanced to adapt to the condition of PCR high-temperature long-time circulation, and PCR is enhanced by the aid of the capacity of catalytic decomposition of pyrophosphoric acid.

Description

technical field [0001] The invention belongs to the field of polymer protein coupling technology and its application in biology, and specifically relates to the synthesis of a novel temperature-responsive inorganic pyrophosphatase, through the modification of a temperature-responsive polymer polyN- Isopropylacrylamide (PNIPAM) is covalently bound to a site-directed mutagenesis inorganic pyrophosphatase (PPase) containing a sulfhydryl group to obtain a conjugate and enhance the efficiency of the polymerase chain reaction (PCR). Background technique [0002] Polymerase chain reaction (PCR) is a biological technology that simulates the DNA replication process in vitro, and it plays a vital role in the fields of modern molecular biology research, biogenetic engineering, and clinical medical testing. However, research has found that during the course of the reaction, a large amount of by-product pyrophosphate ions (P 2 o 7 4- , PPi), the presence of PPi will hinder the reactio...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/10C12Q1/34
CPCC12N9/14C12N15/10C12Q2531/113
Inventor 袁琳陈红崔悦诚王宏炜陈高健刘峰
Owner SUZHOU UNIV
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