Chondrosulphatase AC fusion protein, and coding gene and construction method thereof
A chondroitinase, chondroitin sulfate technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problem of inability to increase protein solubility, difficulty in improving purification effect and protein solubility ratio, weak affinity, etc. question
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Embodiment 1
[0105] Embodiment 1, expression of chondroitin sulfate enzyme AC fusion protein (MBP-ChSase AC)
[0106] 1. Cloning of Flavobacterium heparinus chondroitinase AC coding sequence with signal peptide removed
[0107] The specific process of expression vector pMAL-ChSase AC construction is as follows:
[0108] 1. Design and synthesis of primers
[0109]The DNA sequence of Flavobacterium heparinus chondroitinase AC 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 enzymeschondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum. Appl. Environ. Microbiol. 2000, 6 , 29-35), and then design primers according to the DNA sequence of Flavobacterium heparinus chondroitinase AC that encodes the signal peptide base, and introduce the recognition sites of...
Embodiment 2
[0128] 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 AC fusion protein MBP-ChSase AC
[0129] The optimal expression host E.coli BL21 / pMAL-ChSase AC in Example 1 was selected, and by optimizing the concentration of the inducer IPTG, the expression level of the chondroitinase AC fusion protein MBP-ChSase AC was greatly improved, and the Enzyme activity per unit of fermentation broth.
[0130] Bacterial culture, crushing, protein quantity determination and enzyme activity determination are the same as Step 3 of Example 1. The obtained chondroitinase AC fusion protein can be directly used to measure the activity of chondroitinase AC, and the part of maltose-binding protein is not excised.
[0131] Figure 6 The results of optimizing the IPTG concentration using chondroitin sulfate A or C as a substrate are shown. It can be found that the optimal I...
Embodiment 3
[0133] Embodiment 3, purify chondroitinase AC fusion protein MBP-ChSase AC by amylose column
[0134] The fusion partner maltose-binding protein MBP used in this paper can achieve one-step separation with amylose affinity adsorption. The specific steps of affinity separation are as follows: 100 mL of bacterial cells whose final concentration was 0.24 mM IPTG induced expression for 23 hours were centrifuged at 10,000 rpm for 10 minutes; at the same time, a bacterial cell without induced expression was set as a control. Then proceed with the following two options:
[0135] 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).
[0136] Option 2: Osmotic pressure shock. The bacteria were resuspended in 100 mL osmotic shock buffer I (20-40% sucrose, 30 mM Tris-HCl, 1 mM EDTA) for 15 minutes, and stirred. Centrifuge at 10...
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