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Prokaryotic expression method of human-derived fucose transferase 8 and product of human fucose transferase 8

A fucosyltransferase and prokaryotic expression technology, which is applied in the direction of glycosyltransferase, transferase, and microbial-based methods, can solve the problems of easy degradation and difficult expression and purification, and achieve high biological activity, easy purification, and expression high efficiency effect

Pending Publication Date: 2019-10-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no report of Escherichia coli expressing human Fut8. The main reason is that Fut8 is a mammalian membrane protein resident in the Golgi apparatus. It has glycosylation modification, and it is easy to degrade in the prokaryotic system, making it difficult to express and purify.

Method used

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  • Prokaryotic expression method of human-derived fucose transferase 8 and product of human fucose transferase 8
  • Prokaryotic expression method of human-derived fucose transferase 8 and product of human fucose transferase 8
  • Prokaryotic expression method of human-derived fucose transferase 8 and product of human fucose transferase 8

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Prokaryotic expression and purification of human Fut8ΔTM

[0030] According to the analysis of the protein structure in the UniPort database, the N-terminal transmembrane domain of Fut8 was truncated (Fut8ΔTM, aa68-545), and the prokaryotic expression vector pET28a-Fut8ΔTM was constructed. Transform the recombinant prokaryotic expression plasmid into ROSETTA prokaryotic expression host bacteria, spread it on LB+kanamycin+chloramphenicol (34μg / mL) plate, pick a single colony from the transformation plate the next day, and inoculate it on 5mL LB+kanamycin cultured overnight at 37°C in a chloramphenicol + chloramphenicol liquid medium. Inoculate 2 mL of the overnight cultured bacterial solution into 200 mL of TB+kanamycin+chloramphenicol liquid medium, shake at 37°C for 3 hours at 200 r / min, and make the OD 600After reaching 0.6-0.8, the temperature was lowered to 16° C. and cultured for 1 hour, then IPTG with a final concentration of 0.1 mM was added, and the ...

Embodiment 2

[0033] Example 2: In vitro activity detection of Fut8ΔTM

[0034] The standard enzyme activity detection system was as follows (50 μL): 100 mM MES / NaOH (pH 6.0), 0.1 μM Fmoc-Asn-Gn2Man3Gn2, 0.1 mM GDP-L-Fucose, 20 μg / mL Fut8ΔTM, and the reaction system was incubated at 37 ° C for 5 h. After the reaction, the reaction system was centrifuged at 15,000 r / min for 5 min, and the supernatant was taken for detection by high-performance liquid chromatography, and the reaction system was compared with a standard sample.

[0035] 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 fluorescence detector is 260nm, and the ...

Embodiment 3

[0037] Example 3: Detection of Enzyme Activity of Fut8ΔTM

[0038] The enzyme activity detection system was as follows (50 μL): 100 mM MES / NaOH (pH 6.0), 0.1 μM Fmoc-Asn-Gn2Man3Gn2, 0.1 mM GDP-L-Fucose, 20 μg / mL Fut8ΔTM, and the reaction system was incubated at 37 ° C for 45 min. After the reaction, the reaction system was centrifuged at 15,000 r / min for 5 min, and the supernatant was taken for detection by high-performance liquid chromatography, and the reaction system was compared with a standard sample.

[0039] The results showed that the substrate conversion rate was 38.75%, and the enzyme activity was 36IU.

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Abstract

The invention discloses a prokaryotic expression method of a fucose transferase 8 and a product of the fucose transferase 8. The prokaryotic expression method of the fucose transferase 8 comprises thesteps that a truncated fucose transferase 8 is constructed, and Fut8 delta TM is obtained; a recombinant expression plasmid is constructed; expression host bacteria are transformed by the recombinant expression plasmid, and expression and purification treatment is conducted; an amino acid sequence of the Fut8 delta TM is shown in SEQ ID NO:1; the fucose transferase 8 with prokaryotic expressioncomprises an amino acid sequence of the core zone of the fucose transferase 8, and the amino acid sequence of the core zone of the fucose transferase 8 is shown in SEQ ID NO:1. According to the prokaryotic expression method, escherichia coli is utilized for successfully expressing the human-derived truncated Fut8 (Fut8 delta TM), lots of protein can be prepared in a short time and keep high biological activity, and the method has the advantages that the expression efficiency is high, little impure protein is generated, purification is easily conducted, and the cheap and convenient purpose is achieved.

Description

technical field [0001] The invention belongs to the technical field of prokaryotic expression, and in particular relates to a prokaryotic expression method of human fucosyltransferase 8 and a product thereof. Background technique [0002] Fucosylation is a common process of post-translational modification of glycoproteins. Core fucosylation is the addition of an N-acetylglucosamine to the first N-acetylglucosamine at the reducing end of the core pentasaccharide structure (Gn2Man3) of the N-glycan chain. α1-6 linked fucose, which is a fucosylation reaction that occurs in the Golgi apparatus. Previous studies have shown that core fucosylated glycoproteins are particularly abundant in brain tissue, and core fucosylation is closely related to the development of tumors, making the study of core fucosylation by biological and medical workers enduring. The glycosyltransferase that catalyzes the core fucosylation process is fucosyltransferase Fut8, which is a type II membrane prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/70C12R1/19
CPCC12N9/1051C12N15/70C12Y204/01068
Inventor 高晓冬陆天天王宁
Owner JIANGNAN UNIV