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A method for expressing and preparing udp-glucose-hexose-1-phosphate uridine acyltransferase

A uridine phosphate and UDP-technology, which is applied in the field of genetic engineering, can solve the problems of unsuitability for large-scale preparation, low protein expression, cumbersome and time-consuming operations, etc., and achieves low cost, simple and fast cultivation, and high application. effect of value

Active Publication Date: 2021-08-24
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, UDP-glucose-hexose-1-phosphate uridine acyltransferase has a very low yield when extracted from wild bacteria, is difficult to separate and refine, and is not suitable for large-scale preparation
UDP-glucose-hexose-1-phosphate uridine acyltransferase is expressed in Escherichia coli, but it is expressed intracellularly in Escherichia coli, and it is easy to form inclusion bodies. At the same time, the acquisition of the enzyme protein requires the destruction of the cells. The content of miscellaneous proteins is high, separation and purification are difficult, the operation is cumbersome and time-consuming, and the protein expression level is not high

Method used

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  • A method for expressing and preparing udp-glucose-hexose-1-phosphate uridine acyltransferase
  • A method for expressing and preparing udp-glucose-hexose-1-phosphate uridine acyltransferase
  • A method for expressing and preparing udp-glucose-hexose-1-phosphate uridine acyltransferase

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Experimental program
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Effect test

Embodiment 1

[0023] (1) Construction of recombinant expression vector pHT43-GalT

[0024] UDP-glucose-hexose-1-phosphate uridine acyltransferase (GalT) gene (sequence shown in SEQ ID NO: 1) is derived from Bifidobacterium longum JCM 1217, and after PCR amplification and purification, it is connected to the cloning vector pMD19, The recombinant plasmid pMD19-GalT was constructed.

[0025] The recombinant plasmid pMD19-GalT and the expression vector pHT43 were double-digested with XbaI and KpnI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pHT43-GalT.

[0026] The recombinant expression vector pHT43-GalT was transformed into Bacillus subtilis WB800N, spread on LB plates containing chloramphenicol (5ug / mL) resistance, cultured overnight at 37°C, picked transformants, extracted recombinant plasmids and verified by double enzyme digestion. Such as figure 2 As shown, there are two fragments after digestion, the sizes are about 8000bp (expression vecto...

Embodiment 2

[0043] (1) Construction of recombinant expression vector pHT43-GalT

[0044] The recombinant plasmid pMD19-GalT prepared in Example 1 and the expression vector pHT43 were double digested with XbaI and KpnI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pHT43-GalT.

[0045] The recombinant expression vector pHT43-GalT was transformed into Bacillus subtilis WB800, spread on LB plate containing chloramphenicol (5ug / mL) resistance, cultured overnight at 37°C, picked transformants, extracted recombinant plasmids and verified by double enzyme digestion. Such as figure 2 As shown, there are two fragments after digestion, the sizes are about 8000bp (expression vector pHT43) and 2637bp (UDP-glucose-hexose-1-phosphate uridine acyltransferase), respectively, indicating that the connection is successful.

[0046] (2) Recombinant engineering bacteria

[0047] The constructed recombinant expression vector pHT43-GalT was transformed by electric sho...

Embodiment 3

[0053] (1) Construction of recombinant expression vector pMA5-GalT

[0054] The recombinant plasmid pMD19-GalT and the shuttle vector pMA5 were prepared according to Example 1, which were double-digested with XbaI and KpnI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pMA5-GalT.

[0055] The recombinant expression vector pMA5-GalT was transformed into Bacillus subtilis 168, coated with LB plates containing ampicillin (100ug / mL) resistance, cultured overnight at 37°C, the transformants were picked, and the recombinant plasmid was extracted and verified by double enzyme digestion. The build was successful.

[0056] (2) Recombinant engineering bacteria

[0057] The constructed recombinant expression vector pMA5-GalT was transformed by electric shock. Bacillus subtilis 168 electroporated competent cells were mixed with the recombinant expression vector pMA5-GalT plasmid, and then electric shock was performed after adding to the electric s...

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Abstract

The invention relates to a method for efficiently expressing and preparing UDP-glucose-hexose-1-phosphate uridyltransferase, which is to combine UDP-glucose-hexose-1-phosphate uridylyltransferase (GalT) gene with Bacillus subtilis Bacillus vectors are used to construct recombinant expression vectors. Then the recombinant expression vector was transformed into Bacillus subtilis to construct recombinant engineering bacteria. The recombinant engineered bacteria were induced and cultivated in the liquid medium, the bacterial liquid was centrifuged, and the supernatant was taken. The method UDP-glucose-hexose-1-phosphate uridine acyltransferase yield of the present invention is high, and protein is relatively pure, and recovery purification is easy, and production operation is simple, provides convenience for the industrialized large-scale production of GalT, improves yield, saves Save time and effort, save cost. In particular, the application of the enzyme in the food industry provides a safety guarantee, which is of great significance.

Description

technical field [0001] The invention relates to a recombinant engineering bacterium capable of efficiently expressing UDP-glucose-hexose-1-phosphate uridine acyltransferase and a method for preparing UDP-glucose-hexose-1-phosphate uridine acyltransferase by improving high-efficiency expression , belongs to the technical field of genetic engineering. Background technique [0002] UDP-glucose-hexose 1-phosphaturidylyltransferase (UDP-glucose-hexose 1-phosphaturidylyltransferase, GalT), also known as hexose-1-phosphate uridylyltransferase, or galactose-1-phosphaturia Glycolyltransferase is a protease that catalyzes the conversion between UDP-glucose, galactose-1-phosphate and UDP-galactose, glucose-1-phosphate encoded by the galt gene. Among them, glucose-1-phosphate is the main energy source of most cells, and UDP-galactose is an important component of proteins and fats. This modified protein plays a role in chemical signals, building cell structures, transferring small molec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12N1/21C12N9/12C12R1/125
CPCC12N9/1241C12N15/75C12Y207/07012
Inventor 李拖平李苏红杨强佟超男孙玥
Owner SHENYANG AGRI UNIV
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