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Preparation method of recombinant coral snake poison protein MitTx

A technology of toxin protein and coral snake, applied in the biological field, can solve problems such as complex process, difficult synthesis and folding, complex components, etc., and achieve the effect of increasing protein expression, saving purification cost, and improving purification efficiency

Inactive Publication Date: 2015-05-20
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

The process of extracting MitTx from coral snake venom is complex, expensive, coupled with low protein yield and complex components, which greatly restrict scientific research and application
Moreover, MitTx, especially the β subunit, has a large number of cysteines, and all of them form disulfide bonds, which makes it very difficult to synthesize and fold

Method used

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  • Preparation method of recombinant coral snake poison protein MitTx
  • Preparation method of recombinant coral snake poison protein MitTx
  • Preparation method of recombinant coral snake poison protein MitTx

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0043] 1. Optimized synthesis of MitTx coding gene and construction of recombinant expression plasmid.

[0044] According to the amino acid sequence of MitTxα and β subunits ( figure 1 , figure 2 ), the complete sequence was optimized and synthesized by Nanjing GenScript Biotech Co., Ltd. according to the preferred codons of Escherichia coli.

[0045] The target gene was excised from pUC57 by enzyme digestion technology, pET22b was double-digested with NdeI and XhoI, and then MitTxα-pET22b and MitTxβ-pET22b recombinant vectors were constructed using T4 DNA ligase and recombination technology.

[0046] Pre-expression analysis of recombinant expression vectors: Pick a single transformed colony and culture it in 5 mL of 2×YT medium containing 100 μg / mL Ampicillin until OD 600 When ≈0.6, add IPTG to 1mM, continue to culture for 3h to induce protein expression, and analyze by SDS-PAGE to select high-expressing clones (such as Figure 4 shown).

[0047] 2. Renaturation of MitT...

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Abstract

The invention discloses a preparation method of recombinant coral snake poison protein MitTx. The preparation method comprises the following steps: (1) optimizing gene sequences of MitTxahpha and MitTxbeta; (2) carrying out expression and renaturation on the proteins of MitTxahpha and MitTxbeta; (3) carrying out cation exchange and purification on renatured MitTxahpha; (4) carrying out anion exchange and purification on renatured MitTxbeta; (5) exchanging and purifying cations of a breakthrough product in the step (3); and (6) carrying out binding experiment on products of the steps (2) and (4). According to the preparation method, a pure MitTx protein product can be obtained; and the preparation method can be applied to large-scale production and purification, so that the purification cost is saved, and the purification efficiency is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of recombinant coral snake toxin protein MitTx. In this method, the MitTx protein coding sequence is optimized, and Escherichia coli is used for recombinant expression, and the expressed protein is refolded and purified to obtain a recombinant protein with biological activity. Background technique [0002] MitTx was isolated from the venom of the Texas coral snake by Bohlen in 2011. It consists of α and β subunits, which can interact in a 1:1 non-covalent manner (Kd=12.2±3.1 nM). Among them, the α subunit consists of 61 amino acids, contains 3 pairs of disulfide bonds, and has a molecular weight of 7.26kD; the β subunit contains 126 amino acids, contains 7 pairs of disulfide bonds, and has a molecular weight of 14.50kD (see image 3 ). The two subunits themselves cannot activate sensory neurons, and when the two subunits are combined, intracellular ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/12C07K14/46
Inventor 李俊徐江陈永恒陈林闫宁宁陈主初
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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