Chondrosulphatase B fusion protein, and coding gene and construction method thereof

A chondroitinase and chondroitin sulfate technology, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of weak affinity with the affinity carrier, inability to increase protein solubility, and difficulty in improving the protein solubility ratio of purification effect.

Active Publication Date: 2013-05-15
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, His-tag has obvious disadvantages, that is, the affinity with the affinity carrier is not strong, the solubility of the protein bound to it cannot be increased, and it is difficult to improve the purification effect and protein solubility ratio, etc.
In Pojasek's paper, there is no clear report on the purification effect of His-tag and the soluble ratio of the protein (see Kevin Pojasek, Zachary Shriver, Patrick Kiley, Ganesh Venkataraman and Ram Sasisekharan. Recombinant Expression, Purification and Kinetic Characterization of Chondroitinase AC and Chondroitinase B from Flavobacterium hparinum. Biochemical and Biophysical Research Communications, 2001, 286: 343-351)

Method used

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  • Chondrosulphatase B fusion protein, and coding gene and construction method thereof
  • Chondrosulphatase B fusion protein, and coding gene and construction method thereof
  • Chondrosulphatase B fusion protein, and coding gene and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0104] Embodiment 1, expression of chondroitin sulfate B fusion protein MBP-ChSase B

[0105] 1. Cloning of Flavobacterium heparinus chondroitinase B coding sequence with signal peptide removed

[0106] The specific process of the construction of the expression vector pMAL-ChSase B is as follows:

[0107] 1. Design and synthesis of primers

[0108] The DNA sequence of Flavobacterium heparinus chondroitinase B was queried through Genbank (Tkalec, A.L., Fink, D., Blain, F., Zhang-Sun, G., Laliberte, M., Bennett, D.C., Gu, K. , Zimmermann, J.J. and Su, H. Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum.Appl.Environ.Microbiol6.2000, (1), 29-35), and then design primers according to the DNA sequence of Flavobacterium heparinus chondroitinase B that encodes the signal peptide base, and introduce the recognition sites of res...

Embodiment 2

[0127] Embodiment 2, by optimizing the concentration of the inducer IPTG and the composition of the medium to improve the softness of sulfate Expression and activity of osteosinase B fusion protein MBP-ChSase B

[0128] The optimal expression host E.coli TB1 / pMAL-ChSase B in Example 1 was selected, and by optimizing the concentration of the inducer IPTG, the expression level of the chondroitinase B fusion protein MBP-ChSase B was greatly improved, and the Enzyme activity per unit of fermentation broth.

[0129] Bacterial culture, crushing, protein quantity determination and enzyme activity determination are the same as Step 3 of Example 1. The obtained chondroitin sulfate B fusion protein can be directly used to measure the activity of chondroitin sulfate B, and the part of maltose binding protein is not excised.

[0130] Image 6 The results of optimization of the inducer IPTG concentration are shown. It can be found that the optimal IPTG concentration for inducing the...

Embodiment 3

[0132] Example 3, Purification of Chondroitinase B Fusion Protein MBP-ChSase B by Amylose Column

[0133] The fusion partner (fusion partner) maltose binding protein MBP utilized in the present invention can achieve one-step separation with amylose affinity adsorption. The specific steps of affinity separation are as follows: 100 mL of bacterium whose final concentration was 0.24 mM IPTG induced expression for 23 hours was centrifuged at 10,000 rpm for 10 minutes; at the same time, a bacterium with no induced expression was set as a control. Then proceed with the following two options:

[0134]Option 1: Wash twice with Column buffer (20mM Tris-HCl, 200mM NaCl, pH7.5), resuspend in 5mL Column buffer, and perform sonication (300W output power, 3 seconds each time and intermittent 99 times in 3 seconds).

[0135] Option 2: Osmotic pressure shock. Resuspend the bacteria in 100 mL osmotic shock buffer I (20-40% sucrose, 30 mM Tris-HCl, 1 mM EDTA) for 15 minutes, and stir. Cen...

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Abstract

The invention discloses a chondrosulphatase B fusion protein, and a coding gene and a construction method thereof. The chondrosulphatase B fusion protein comprises maltose-binding protein. The invention also provides a method for purifying the chondrosulphatase B fusion protein. Furthermore, the invention also relates to a method for producing chondroitin sulfate B of low molecular weight.

Description

technical field [0001] The invention relates to a chondroitinase B fusion protein, its coding gene and its construction method in the field of genetic engineering and fermentation engineering. In addition, the present invention also provides a method for purifying the chondroitinsulfate B fusion protein. In addition, the present invention also relates to a method for producing low molecular weight chondroitin sulfate B. Background technique [0002] Chondroitin sulfate lyase (chondroitinase or chondroitin sulfaeyase, hereinafter sometimes referred to as "ChSase") is a kind of glycosaminoglycans that can degrade chondroitin sulfate, chondroitin, hyaluronic acid and other glycosaminoglycans into unsaturated disaccharides (ΔDi and oligosaccharides). Sugar lyase. In recent years, ChSase has been paid more and more attention to as a tool for studying the structure and function of proteoglycans in the body and as a new type of medicinal enzyme. Chondroitinase B (hereinafter somet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/62C12N15/63C12N1/21C12N1/15C12N1/19C12N5/10C12P19/04
CPCY02P20/52
Inventor 吴敬君李晔邢新会张翀
Owner TSINGHUA UNIV
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