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Botulinum neurotoxin protein crystal structure and crystal preparation method and determination method

A technology of botulinum toxin, crystal structure, applied in biological field

Inactive Publication Date: 2020-09-18
四川一埃科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, obtaining crystals of the functional domain of BONT and analyzing its three-dimensional structure is worthy of in-depth study, but it has not been reported yet in China

Method used

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  • Botulinum neurotoxin protein crystal structure and crystal preparation method and determination method
  • Botulinum neurotoxin protein crystal structure and crystal preparation method and determination method
  • Botulinum neurotoxin protein crystal structure and crystal preparation method and determination method

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Experimental program
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Embodiment 1

[0035] The preparation of embodiment 1 botulinum toxin protein crystal

[0036] Due to the complex structure of the wild-type BONT protein and its great toxicity, the amino acid sequence provided by the present invention is a functional fragment of the functional domain of the BONT protein specifically selected by the inventor (with a biologically active fragment of the BONT protein), which can not only A large amount of recombinant expression in bacilli can also obtain recombinant proteins with biological activity.

[0037] The functional fragment of the BONT protein functional domain selected in the present invention and the amino acid sequence of the BONT protein functional domain are as shown in SEQ ID NO: 1, specifically:

[0038] MKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIE VILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLN YGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGR LIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLY DNQSNSGILKDFWGDYLQYDKP...

Embodiment 2

[0055] Example 2 Screening and structural analysis of botulinum toxin protein functional domain crystals

[0056] Concentrate the purified BONT protein to 12mg / ml, mix with buffer pH 7.6, 20mM Tris-HCl and 0.5M NaCl, incubate on ice for 1h, centrifuge at 12000r / min for 30min at 4°C, and use the sitting drop method initial screening. The growth conditions are as follows: add 500ul of pool solution to each well of the seat drop plate, add growth droplets containing 1 μL of protein complex and 1 μL of pool solution to the crystal wells, culture at 20°C for 1 week, and observe with a polarizing microscope . Optimizing the conditions for better crystal growth, the pendant drop method was used to optimize the crystal conditions until a single crystal with better crystal shape and strong diffraction ability was obtained (attached Figure 4 ). The conditions of the final crystallization pool solution were 0.1M Hepes (pH7.5), 12%polyethylene glycol 8000, 8% glycerol and 100mM NaCl. ...

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Abstract

The invention provides a botulinum neurotoxin protein crystal structure and a crystal preparation method and determination method. The crystal has a space group P2<1>2<1>2<1>, and crystal cell parameters are as shown in the description. Through a genetic engineering method, BONT proteins being high in purity can be successfully expressed and purified, and besides, a crystallizing condition that the proteins can form perfect crystal forms can be screened; and finally, through X ray crystal diffraction, crystal data is collected, and the crystal structure of the proteins is further determined. Afoundation is provided for medicine design and an associated molecular action mechanism based on the protein structure in the future.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a botulinum toxin protein crystal structure, a crystal preparation method and an analysis method. Background technique [0002] Botulinum neurotoxin (BoNT), a neurotoxin produced by the anaerobic Clostridium botulinum, is the most toxic protein toxin known so far [1-3] . Botulinum toxin is divided into 7 types of serotypes (A-G), the most common of which is type A. Based on current research, BoNT has been applied to medical cosmetology and the treatment of migraine, eye motility, movement disorders and other diseases [4-5] . Relevant studies have pointed out that at the neuromuscular junction, botulinum toxin type A (BoNT / A) can tightly bind to receptors on the surface of nerve cells, and send the light chain into the cell through the transmembrane with the help of the N-terminal of its heavy chain, and then Blocking acetylcholine release by cleavage of SNAP25, a synap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/33C07K1/22C07K1/30C12N15/70G16B15/20
CPCC07K14/33C07K2299/00C12N15/70G16B15/20
Inventor 唐麟
Owner 四川一埃科技有限公司
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