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Multi-copy metallothionein recombinant expression vector and method thereof for high-efficiency expression of metallothionein

A technology of metallothionein and expression vectors, applied in the direction of microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of cumbersome purification steps, consumption of animal resources, large investment in equipment, etc., and achieve cumbersome purification steps , optimization of fermentation conditions and high production cost

Inactive Publication Date: 2014-07-02
山东东兴生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using this method to obtain metallothionein requires large investment in equipment, high transfer costs, cumbersome purification steps, and extremely low yield (up to 8.6mg / g pig liver), which requires the consumption of a large amount of animal resources
At present, there have been reports in the literature on the use of genetic engineering to construct a method for the production of metallothionein by Escherichia coli engineering bacteria, but the yield is low (CN1141395C)
Although fusion protein expression technology provides a new method for the expression of metallothionein (CN101144093B), it still has the disadvantage of low yield (up to 25.2mg / L fermentation broth)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The steps of the method for efficiently expressing metallothionein are:

[0029] (1) Construction of recombinant expression vector

[0030] The metallothionein coding sequence was redesigned and synthesized according to Pichia pastoris codon preference. Then, the metallothionein coding sequence was amplified by PCR, and the gene was inserted into the EcoRI site on the vector pAO815 through restriction enzyme digestion and ligation. , Constructed into an expression vector containing a single copy metallothionein expression cassette (pAOX1 and metallothionein coding sequence);

[0031] Isolate the metallothionein expression cassette from the expression vector constructed above with restriction enzymes BglII and BamHI, and insert it again into the BamHI site of the above expression vector to obtain an expression vector in which two metallothionein expression cassettes are connected in series , Repeat the insertion procedure to construct a series of recombinant expression vectors...

Embodiment 2

[0039] The optimization steps of this method for efficiently expressing metallothionein are:

[0040] (1) Construction of recombinant expression vector

[0041] First, the metallothionein coding sequence was redesigned and synthesized according to the Pichia pastoris codon preference, and then the metallothionein coding sequence was amplified by PCR, and the gene was inserted into the EcoRI site on the vector pAO815 through restriction digestion and ligation. Then, transform competent E. coli Top10F', select Amp resistant clones on LB plates containing 50ug / ul Amp, pick Amp resistant transformants, inoculate them in a medium containing 50ug / ul Amp, and cultivate overnight at 37°C with shaking , Extract plasmid DNA;

[0042] Use the above-mentioned plasmid DNA as a template and 5’AOX1 and 3’AOX1 as sequencing primers to perform sequencing to verify the correct insertion of the target gene;

[0043] The metallothionein expression cassette (pAOX1 and metallothionein coding sequence) was...

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Abstract

The invention provides a multi-copy metallothionein recombinant expression vector and a method thereof for high-efficiency expression of metallothionein, and belongs to the technical field of biological chemistry. The method is as follows: constructing the multi-copy metallothionein recombinant expression vector; converting the constructed multi-copy metallothionein recombinant expression vector in pichia pastoris GS115 or KM71, and screening on a histidine-free culture medium to obtain recombinant bacteria; inoculating the screened recombinant bacteria in a BMGY culture solution, rapidly oscillating for culture, centrifugally collecting bacteria, resuspending with the culture solution, diluting, rapidly oscillating for culture until log phase, adding methanol, and feeding the methanol in batches to continue to cultivate; collecting the bacteria, ultrasonically crushing, collecting supernatant by centrifugation, and separating and purifying the supernatant to obtain the metallothionein. The method overcomes the defects of a traditional metallothionein preparation method, can effectively avoid the waste of a lot of animal resources and bacteria sourced endotoxin contamination, reduces the production cost, and improves the protein yield.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a multi-copy metallothionein recombinant expression vector and a method for expressing metallothionein with high efficiency. Background technique [0002] Generally, Metallothioneins (MTs for short) are a class of low-molecular-weight proteins that are rich in sulfhydryl groups and have a high affinity with metal ions. Metallothionein is widely distributed in the biological world. It contains 61 amino acids, 20 of which are cysteine, and a few have 21. Each molecule can bind 7-12 metal ions. Because MTs molecules are rich in sulfhydryl groups, they have a variety of important physiological functions in organisms, such as detoxification of heavy metals, balance adjustment of trace elements in the body, and scavenging of free radicals. Due to its broad-spectrum and high-efficiency health care effects and high medical value, it can be applied to medicine, food health care and ...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12R1/84
Inventor 唐东起崔泰兴吴琦葛瑞蕾赵亚茹
Owner 山东东兴生物科技股份有限公司
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