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Method for increasing soluble expression of recombinant GnT-II (N-acetylglucosaminyl transferase II)

A technology of acetylglucosamine and transferase, applied in the direction of acyltransferase, transferase, recombinant DNA technology, etc., can solve the problems of low success rate, difficult soluble expression, and complicated steps.

Active Publication Date: 2020-08-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this process is cumbersome and the success rate is low
At present, recombinant truncated human GnT-II (GnT-II-ΔTM) has been successfully expressed and active in E. coli, but most of the produced proteins are inclusion bodies. How to increase its soluble expression level is a difficult point in this field.

Method used

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  • Method for increasing soluble expression of recombinant GnT-II (N-acetylglucosaminyl transferase II)
  • Method for increasing soluble expression of recombinant GnT-II (N-acetylglucosaminyl transferase II)
  • Method for increasing soluble expression of recombinant GnT-II (N-acetylglucosaminyl transferase II)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Induced expression of GnT-II-ΔTM-Trx protein

[0041] 1. Obtain the recombinant prokaryotic expression strain of GnT-II-ΔTM-Trx

[0042] Select a single colony with successful sequencing and inoculate it into 50 μg / mL kanamycin liquid medium, culture overnight at 37°C and 200 rpm, and extract the pET28a-GnT-II-ΔTM-Trx recombinant expression vector according to the Sangon plasmid mini-extraction kit out, the recombinant expression vector was transformed into Escherichia coli Rosetta (DE3) and Rosetta gami2 (DE3) strains, and the expression of GnT-II-ΔTM-Trx protein was detected. Transformation process: Take 1uL of each plasmid (100ng / uL) and transfer it into 50μL of Rosetta (DE3) and 50μL of Rosetta gami2 (DE3) competent cells, incubate on ice for 30min, heat shock at 42°C for 1min, transfer to ice for 2min, Finally spread on solid plates containing the desired antibiotics.

[0043] 2. Cultivate the activated strain overnight

[0044] The above-mentioned re...

example 2

[0047] Example 2: In vitro activity detection of GnT-II-ΔTM-Trx

[0048] The standard enzyme activity detection system is as follows (50μL): 100mM MES / NaOH (pH 6.0), 10mM MnCl 2 , 0.2μM Fmoc-GlcNAc2Man3, 0.2mM UDP-GlcNAc, 20μg / mL GnT-I-ΔTM, 200μg / mL GnT-II-ΔTM, and the reaction system was incubated at 37°C for more than 5h. After the reaction, the reaction system was centrifuged at 18,000 g for 5 min, and the supernatant was taken for detection by high performance liquid chromatography, and the reaction system was compared with the standard sample.

[0049] The detection conditions of high-performance liquid chromatography are as follows: the instrument used is high-performance liquid chromatography Alliance e2695HPLC (Waters), the fluorescence detector used is 2475FLR Detector (Waters), and the liquid chromatography column used is an amino column (TOSHO TSKgel Amide-80 3 μm 4.6× 150mm), wherein the Fmoc-labeled sugar chain is detected, the excitation wavelength used by the f...

example 3

[0050] Example 3: Optimization of GnT-II-ΔTM-Trx protein-induced expression conditions

[0051] 1. Obtain the recombinant expression strain of GnT-II-ΔTM-Trx

[0052] Select the single clone successfully sequenced in Example 1 and inoculate it into 50 μg / mL kanamycin liquid medium, culture overnight at 37°C and 200 rpm, and recombine according to the Shengong plasmid mini-extraction kit pET28a-GnT-II-ΔTM-Trx The expression vector was extracted, and the recombinant expression vector was transformed into Escherichia coli Rosetta (DE3) and Rosetta gami2 (DE3) strains, and spread on a medium containing 50 μg / mL kanamycin + 34 μg / mL chloramphenicol (Rosetta gami2 (DE3) supplemented 50 μg / mL tetracycline + 50 μg / mL streptomycin) plate for overnight culture, pick a single colony and inoculate it on a plate containing 50 μg / mL kanamycin + 34 μg / mL chloramphenicol (Rosettagami2 (DE3) plus 50 μg / mL tetracycline +50μg / mL streptomycin) liquid medium, culture overnight at 37°C, 200rpm, ad...

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Abstract

The present invention discloses a method for increasing soluble expression of recombinant GnT-II (N-acetylglucosaminyl transferase II). The method for increasing the soluble expression level of the recombinant GnT-II in Escherichia coli comprises the steps as follows: constructing a truncated GnT-II to obtain GnT-II-deltaTM with sequence as shown in SEQ ID NO:1; constructing a recombinant expression plasmid; transforming the recombinant expression plasmid into an expression host bacteria to construct a recombinant prokaryotic expression strain; culturing the recombinant prokaryotic expressionstrain to induce expression. The GnT-II is successfully expressed in the Escherichia coli, the soluble expression level of the recombinant human GnT-II is increased, the GnT-II has catalytic activityin vitro, the technical problems that mammalian membrane protein GnT-II is easy to degrade and difficult to purify are solved, and the recombinant GnT-II can be prepared in large quantities.

Description

technical field [0001] The invention belongs to the technical field of soluble expression of recombinant N-acetylglucosamine transferase II, and specifically relates to a method for improving the soluble expression of recombinant N-acetylglucosamine transferase II. Background technique [0002] Glycosylation refers to the process of modifying sugars (oligosaccharides) on proteins or lipids to form sugar complexes, and is one of the main forms of post-translational modification in eukaryotic cells. The N-glycosylation modification of protein directly affects the structure and function of protein, which has important physiological significance. There are various forms of N-oligosaccharides, and the biosynthetic pathways of N-oligosaccharides are involved in various glycosyltransferases. Therefore, the preparation and properties of glycosyltransferases are one of the important directions in the field of glycoscience. N-acetylglucosamine transferase II (GnT-II) is one of the ke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/70
CPCC12N9/1029C12Y203/01003C12N15/70
Inventor 高晓冬项梦海王宁陆天天
Owner JIANGNAN UNIV