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Preparation method and application of chondroitin sulfate ABC enzyme fusion protein

A chondroitin sulfate, fusion protein technology, applied in the directions of lyase, oxidoreductase, recombinant DNA technology, etc., can solve the problems of cumbersome preparation process, cumbersome extraction steps, limited research on heterologous recombinant expression, etc., and achieves simplified purification procedures, Generate low-quality effects

Active Publication Date: 2016-03-30
BEIJING POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the research on the heterologous recombination expression of ChSaseABC is very limited. In the prior art, the proportion of ChSaseABC enzyme protein expressed in the active protein state after using the prokaryotic expression system is very low. The denatured protein needs to undergo denaturation and renaturation process, and the preparation process is cumbersome.
[0008] In addition, the patent application CN103305496A discloses a method for extracting chondroitin sulfate from microorganisms, but the extraction steps of this method are cumbersome and the cost is high

Method used

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  • Preparation method and application of chondroitin sulfate ABC enzyme fusion protein
  • Preparation method and application of chondroitin sulfate ABC enzyme fusion protein
  • Preparation method and application of chondroitin sulfate ABC enzyme fusion protein

Examples

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Embodiment 1

[0093] Embodiment 1, the expression of chondroitin sulfate enzyme ABC fusion protein (GAPDH-ChSaseABCI) Express

[0094] 1. Cloning of Proteus vulgaris chondroitin sulfate ABC enzyme coding sequence and glyceraldehyde 3-phosphate dehydrogenase DNA sequence without signal peptide

[0095] The specific process of constructing the expression vector pMAL-c2x-ChSaseABCI is as follows:

[0096] 1.1 Design and synthesis of primers

[0097] The DNA sequences of Proteus vulgaris chondroitin sulfate ABC enzyme and glyceraldehyde 3-phosphate dehydrogenase were obtained through Genbank query, and the upstream and downstream primers used were:

[0098] Upstream primer P1: 5'-CG GGATCC ATGGCCACCAGCAATCCTGCATT-3' (SEQ ID NO: 5) (the underlined base is the restriction site of BamHI),

[0099] Downstream primer P2: 5'-AA CTGCAG TTATCAAGGGAGTGGCGAGAGTTTG-3' (SEQ ID NO: 6) (the underlined base is the PstI restriction site), after amplification, the BamHI and PstI restriction sites were...

Embodiment 2

[0127] Embodiment 2, purify chondroitin sulfate ABC enzyme fusion protein by Ni column GAPDH-ChSaseABCI

[0128] In this example, a Ni column is used to realize affinity adsorption to realize one-step separation.

[0129] The specific steps of affinity separation are as follows: 50 mL of the bacterial cells whose final concentration was 0.5 mMIPTG induced expression of 24 were centrifuged at 6000 rpm for 10 minutes; at the same time, the bacterial cells without induced expression were set as a control. Then proceed with the following two options:

[0130] Wash twice with column equilibration solution Columnbuffer (20mM Tris-HCl, 200mMNaCl, pH7.4), resuspend in 30mL Columnbuffer, carry out sonication (output power is 300W, each sonication 5 seconds and interval 6 seconds, the total processing time is 15 minute).

[0131] After centrifugation, the supernatant was passed through a 1 mL pre-equilibrated Ni affinity separation column at 1 mL / min, eluted by the eluent and coll...

Embodiment 3

[0144] Embodiment 3, utilize the chondroitin sulfate ABC enzyme that embodiment 2 prepares to produce low molecular weight sulfuric acid Chondroitin A, B or C

[0145] The chondroitin sulfate ABC enzyme with a purity of 90% was obtained in Example 2. This embodiment uses the purified chondroitin sulfate ABC enzyme to produce low molecular weight chondroitin sulfate A, B or C.

[0146] Concrete production method is as follows: preparation concentration is respectively the chondroitin sulfate A of 100g / L, the solution 1L of B and C, and chondroitin sulfate A, B and C molecular weight are 50kDa, derive from bovine cartilage tissue (purchased from Nanjing Odufoni Biotechnology Co., Ltd.), as a reaction substrate, add 10-20ml of purified chondroitinase (13922IU / L). The reaction temperature is 30-55°C, and 15 mL of new enzyme solution is added every 1 hour, and samples are taken to measure the reaction product at the same time, and the reaction time is 5-10 hours.

[0147] Afte...

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Abstract

The invention provides a method for expressing a chondroitin sulfate ABC enzyme fusion protein. The method comprises: 1) separately cloning a chondroitin sulfate ABC enzyme gene and a 3-glyceraldehyde phosphate apodehydrogenase gene, and connecting the genes to ensure that subsequently expressed 3-glyceraldehyde phosphate apodehydrogenase is connected with an N terminal of chondroitin sulfate ABC enzyme protein; 2) recombining the connected chondroitin sulfate ABC enzyme fusion protein gene into an expression vector; 3) converting the expression vector containing the chondroitin sulfate ABC enzyme fusion protein gene into a host cell for expression; and 4) harvesting and purifying expressed chondroitin sulfate ABC enzyme fusion protein. The active chondroitin sulfate ABC enzyme fusion protein obtained by the method accounts for 70% of the total expression protein quantity, so that subsequent purification processes are greatly simplified. The obtained ChSase ABC I fusion protein has high enzyme activity and can be used for producing chondroitin sulfate; moreover, the generation condition of the chondroitin sulfate is low, and the method for generating the chondroitin sulfate is simple and convenient and is easy to control.

Description

technical field [0001] The invention relates to a method for preparing a chondroitin sulfate ABC fusion protein and its application in the field of genetic engineering and fermentation engineering. Background technique [0002] Chondroitin sulfate lyase (chondroitinase or chondroitinsulfateyase, referred to as "ChSase") is a kind of cleavage enzyme that can degrade glycosaminoglycans such as chondroitin sulfate, chondroitin, and hyaluronic acid into unsaturated disaccharides (ΔDi and oligosaccharides). enzyme. [0003] With the in-depth research on ChSase, it is found that chondroitin sulfate ABC (abbreviated as "ChSaseABC") has a wide range of application values. Researchers use ChSaseABC to detect chondroitin sulfate and produce low molecular weight chondroitin sulfate. Bao Lunjun, Yang Jiancheng, etc. used ChSaseABC enzymatic hydrolysis-high performance liquid chromatography to detect hyaluronic acid in shark fins. Using ZORBAX sugar analysis column, the ultraviolet det...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C12N9/88C12N9/02C12N1/21C12P19/26
Inventor 李晔陈振娅辛秀兰陈亮苏东海章宇宁
Owner BEIJING POLYTECHNIC
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