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Prepn of metallothionein

A metallothionein and metal ion technology, applied in fermentation and other directions, can solve the problems of imperfect expression conditions and purification conditions, low expression yield and high cost, and achieve the effects of low cost, good compatibility and improved product purity.

Inactive Publication Date: 2002-05-08
FUDAN UNIV
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AI Technical Summary

Problems solved by technology

Xiong and Cols were equal to 1997 and published the fusion protein of glutathione sulfur transferase (GST) and metallothionein to express metallothionein at the same time, but due to the imperfect and unreasonable expression conditions and purification conditions, the expression yield Still relatively low (about 3-5mg / L fermentation broth)
Hong et al. used Intrein fusion protein technology in 2001 to express metallothionein with a relatively high yield, but it was only used for the expression of wild-type protein, and the cost was relatively high

Method used

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Examples

Experimental program
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Effect test

Embodiment Construction

[0013] Escherichia coli BL21 lacking some proteolytic enzymes was used as fermentation engineering bacteria.

[0014] On metallothionein and its mutant genes, two endonuclease sites, BamHI (5' end) and EcoRI (3' end), were inserted by PCR method, and then assembled on the pGEX-4T-2 plasmid, Constructed into expression plasmids.

[0015] The expression plasmid was transferred into BL21, after small-scale culture, mixed with glycerol solution (65% (V / V) glycerol, 0.1M MgSO 4 , 25mM Tris-HCl, pH 8.0) 1:1 mixed evenly to make glycerol bacteria, stored at -70°C for a long time. Before fermentation, take out the BL21 containing the expression plasmid from -70°C, insert it into fresh 2YT (containing 100 μg / mL ampicillin), culture at 37°C with rapid shaking (270-330rpm) for 7-16 hours, and transfer at a ratio of 1:100 After that, continue to expand the culture at 30~35℃ to A 600 =0.6-1.0, add IPTG to the final concentration of 0.01-1.0mM, continue to cultivate, and then add metal i...

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Abstract

The present invention is one gene engineering process of preparing metallothionein in high yield. The process incldues constituting the expression plasmid or its mutant gene, transferring and fermentation culture in engineering colibacillus, induction and adding metal ion before further culture, collecting thallus; ultrasonic crushing thallus, centrifugation, static adsorption with affinity chromatographic column of glutathione-agarose gel 4B, incision with thrombase on the column, column chromatograhic desalting, gradient salt elution and collecting destination protein. The present inventioncan obtain destination protein with purity over 95 %. Using the said method to prepare 6 mutant proteins of monkey metallothionein I can obtain yield and purity similar to that in wild recombinant protein.

Description

technical field [0001] The invention is a method for efficiently preparing metallothionein by protein engineering. Background technique [0002] Metallothioneins (MTs for short) [0003] The invention relates to a protein engineering preparation method of metallothioneins (Metallothioneins, MTs). Metallothionein is a class rich in sulfhydryl groups (cysteine ​​accounts for about 1 / 3 of the total number of amino acids) and metal ions (containing 6-7 Zn 2+ ) of low molecular weight proteins. Metallothionein is widely distributed in the biological world and is a class of proteins with many important physiological functions, such as regulating the homeostasis of zinc and copper ions, detoxifying heavy metals, and removing active oxygen free radicals in the body, etc. In order to study the structure and function of metallothionein and develop the application of metallothionein, it is necessary to obtain a large amount of metallothionein. A number of methods for the expression...

Claims

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Application Information

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IPC IPC(8): C12P21/02
Inventor 黄仲贤余文浩高原谢毅
Owner FUDAN UNIV
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