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Recombinant scorpion toxin, its soluble expression and purification

An insect toxin, soluble technology, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of inactivity, low yield, strong hydrophobicity of scorpion neurotoxin, etc., and achieve hydrophobicity strong effect

Inactive Publication Date: 2006-08-16
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, at present, it is very difficult to isolate a single scorpion insect toxin component from natural scorpion venom in terms of quality and quantity. Therefore, cloning the insect toxin gene and expressing the protein through genetic engineering has become the preferred solution
However, due to the strong hydrophobicity of scorpion neurotoxin and rich in disulfide bonds, it is easy to form inactive inclusion bodies in engineering bacteria
Most of the recombinant toxins are now obtained by denaturation, but the yield of this method is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 Soluble fusion expression of recombinant scorpion insect toxin rBmK IT

[0028] 1. PCR primer design and BmK IT gene amplification

[0029] According to the cDNA sequence of the scorpion insect toxin BmK IT, and in order to facilitate protein purification, the present invention designs a nucleotide sequence encoding 6 histidines in the downstream primers. The specific primers are designed as follows:

[0030] Upstream: 5'-CTGA G CC ATG G GC AAG AAG AAT GGA TAC GCT-3', where CC ATG G is the Nco I restriction site;

[0031] Downstream: 5'-CGCG CTG CAG TTA ATG ATG ATG ATG ATG ATG ATG ACC AAT TAT TTGGAC GTC-3', where CTG CAG is the PstI restriction site, ATG ATG ATG ATG ATG ATG ATG It is a nucleotide sequence encoding 6 histidines (6×his).

[0032] The template for PCR amplification comes from the recombinant secretory expression vector pExSecI-BmKIT of the cDNA of the mature protein of the contractile insect toxin BmK IT that we have amplified from ...

Embodiment 2

[0045] Embodiment 2, the purification of recombinant scorpion insect toxin rBmK IT

[0046] 1. Ni-NTA affinity chromatography of recombinant scorpion toxin rBmK IT

[0047] Use a refrigerated centrifuge to collect the bacterium by centrifuging the fermented liquid of the engineering bacteria at 4° C. at a speed of 8000 rpm for 15 min, resuspend it with 0.1M pH8.5 phosphate buffer (containing 20% ​​glycerol, pH8.5), and use Ultrasonic disruptor breaks up the cell, after the centrifugation of 12000rpm of the cell breaking liquid, inject the Ni-NTA column with the clarified cell crude extract, fully combine with the column material, wash buffer (containing 200mM imidazole, 20% Glycerol, pH 6.5) to wash the column material, and then leave it at room temperature overnight to induce the self-cleavage activity of intein under the condition of pH 6.5. The next day, continue to wash the Ni-NTA column with 10 times of the column bed volume of washing buffer, and then use 3 times of the...

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PUM

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Abstract

Recombinant-scorpion entomotoxin rBmKIT, its soluble expression and purification are disclosed. The procedure is carried out by solvation expressing for BmK ITcDNA and a protein self-shearing element, fusing nucleotide sequence with 6 histidine coded on 3'end of BmK IT cDNA, breaking fermented broth of engineering bacterium by bacterium, protein extracting, self-shearing by fused protein, affinity chromatographing, concentrating sampler, high-temperature treating, gel filter chromatographing and purifying to obtain final product. It is soluble and simple.

Description

technical field [0001] The invention relates to a recombinant scorpion insect toxin, in particular to a soluble expression of a recombinant scorpion insect neurotoxin rich in disulfide bonds and a separation and purification method thereof. Background technique [0002] East Asian pincer scorpion (Buthus martensii Karsch) is the main scorpion species in my country. It is widely distributed in Northwest China, Mongolia and Korea. Thousands of years ago, scorpion decoction was used to treat stroke and other diseases. In recent years, it has been found that it contains anticancer peptides, antiepileptic peptides, and antibacterial peptides. The active ingredient of scorpion is a biologically active polypeptide, namely scorpion toxin. It is estimated that there are about 100,000 kinds of scorpion toxins, and only 0.02% of them have been found. Most of them are ion channel blockers, which specifically recognize and bind Na on the cell membrane. + 、K + , Cl - , Ca 2+ Plasma c...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K19/00C12N15/09C12N15/12C12N15/62C12P21/02C07K1/14
Inventor 梁爱华许成钢范晓军王伟柴宝峰崔大伟
Owner SHANXI UNIV
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