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Heparin skeleton synthase and encoding gene and application thereof

A technology for coding genes and skeletons, which is applied to heparin skeleton synthase and its coding genes and application fields to achieve the effect of improving transferase activity

Active Publication Date: 2020-01-03
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

No glycosaminoglycan backbone synthase gene from this fungus was reported by searching the database

Method used

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  • Heparin skeleton synthase and encoding gene and application thereof
  • Heparin skeleton synthase and encoding gene and application thereof
  • Heparin skeleton synthase and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Preparation of Heparin Skeleton Synthase GaGlcNAc-T

[0044] (1) Construction of expression strains

[0045] The recombinant vector pET28a-His-GaGlcNAc-T (synthesized by Nanjing GenScript Co., Ltd.) containing the heparin backbone synthase coding gene (SEQ ID NO.1) was transformed into Escherichia coli BL21 (DE3) competent cells to construct recombinant strains, Kanamycin (100 μg / mL) was cultured on the LB plate for 12h, and the transformants were screened (negative control experiment), and activated to obtain GaGlcNAc-T positive transformants; the amino acid sequence of the heparin backbone synthase was as SEQ ID NO.2 shown;

[0046] The recombinant vector pET28a-His-GaGlcNAc-T(N56D) (synthesized by Nanjing GenScript Co., Ltd.) containing the heparin backbone synthase mutant coding gene (SEQ ID NO.3) was transformed into Escherichia coli BL21(DE3) competent cells, Construct recombinant bacterial strain, and obtain GaGlcNAc-T (N56D) positive transformant ac...

Embodiment 2

[0053] Example 2. Activity verification of heparin backbone synthase GaGlcNAc-T

[0054] (1) Verification of GlcNAc transferase activity of heparin backbone synthase GaGlcNAc-T

[0055] Using commercial GlcA-pNP (0.2mM final concentration) as the acceptor substrate, UDP-GlcNAc (0.3mM final concentration) as the donor substrate, the heparin backbone synthase GaGlcNAc-T prepared in Example 1 and The heparin backbone synthase mutant GaGlcNAc-T(N56D) was reacted as a protease, and the reaction system was shown in Table 2; the reaction was placed in a water bath at 37°C for 4 hours, and heated in boiling water for 5 minutes to inactivate the protease to terminate the reaction. After filtering with a 0.22 μm filter membrane, perform HPLC detection according to the method described in Table 1. The pNP group of the monosaccharide acceptor has specific absorption at the ultraviolet detection wavelength of 310 nm, and the flow rate of the mobile phase is 0.5 mL / min.

[0056] Table 2. R...

Embodiment 3

[0062] Example 3 Study on the Properties of Heparin Skeleton Synthase GaGlcNAc-T

[0063] (1) Determination of optimal reaction pH of enzyme in vitro reaction

[0064] The reaction system is shown in Table 2. The heparin backbone synthase GaGlcNAc-T prepared in Example 1 was used as the protease, and the pH of the buffer solution remained unchanged. The Tris-HCl buffer solution was replaced with Tris-HCl with different pH values. / PBS / MES buffer, respectively set 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 10, a total of 9 pH gradient points, and each gradient has three parallel groups. All the other processing conditions are with embodiment 2.

[0065] The result is as Figure 4 As shown, the results show that the heparin backbone synthase GaGlcNAc-T is active in a wide pH range (6.0-8.5), and the optimum pH of the reaction is 6.5.

[0066] (2) Determination of the optimal metal ion for the in vitro reaction of the enzyme

[0067] The reaction system is shown in Table 2. The ...

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Abstract

The invention relates to heparin skeleton synthase and an encoding gene and application thereof. The amino acid sequence of the heparin skeleton synthase is as shown in SEQ ID NO.2, and the nucleotidesequence of the encoding gene is as shown in SEQ ID NO.1. The heparin skeleton synthase is derived from Gallibacterium anatis, and is a functional enzyme for catalyzing glycosyl transfer. A heparin skeleton synthase mutant is obtained by mutating asparagine at the 56th position in the amino acid sequence of the heparin skeleton synthase into aspartic acid, the amino acid sequence of the mutant isshown as SEQ ID NO.4, and the nucleotide sequence of the encoding gene of the mutant is shown as SEQ ID NO.3. The heparin skeleton synthase disclosed by the invention is heparin skeleton synthase with a brand-new source, has transferase activity of acetylglucosamine, and can be used for effectively synthesizing a heparin disaccharide skeleton by utilizing substrates GlcA-pNP and UDP-GlcNA, so that a foundation is laid for further synthesizing a heparin sugar chain.

Description

technical field [0001] The present invention relates to a heparin skeleton synthase, its coding gene and its application, in particular to the heparin skeleton synthase derived from Gallibacterium anatis, its highly active mutant, its coding gene and its application in the synthesis of heparin skeleton , belonging to the field of biotechnology. Background technique [0002] Glycosaminoglycan (Glycosaminoglycan, GAG) is a polyanionic linear polysaccharide formed by regular arrangement of repeating disaccharide units composed of hexuronic acid or hexose and hexosamine. Glycosaminoglycans are widely distributed and various, mainly including hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparan sulfate, heparin, keratan sulfate, etc. In recent years, people have extracted and isolated glycosaminoglycans from bacteria and marine organisms. Almost all glycosaminoglycans contain -NAc, -COOH, -OH, -SO 3 H and other groups, these groups play a vital role in the biological...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/26C12P19/18C12R1/19
CPCC12N9/1051C12N15/70C12P19/26C12P19/18C12Y204/01155
Inventor 生举正凌沛学王田田邓建群
Owner SHANDONG UNIV
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