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Polypeptide of scorpion toxin and synthetic method thereof

A synthesis method and technology of scorpion toxin, applied in the field of scorpion toxin polypeptide and its synthesis, can solve the problems of easy occurrence of mismatch and low renaturation efficiency, and achieve the effects of stable protein spatial conformation, simple operation and high activity.

Active Publication Date: 2017-07-18
合肥科生景肽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for proteins containing multiple pairs of disulfide bonds, mismatches are often prone to occur during the renaturation process, resulting in low renaturation efficiency.

Method used

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  • Polypeptide of scorpion toxin and synthetic method thereof
  • Polypeptide of scorpion toxin and synthetic method thereof
  • Polypeptide of scorpion toxin and synthetic method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Preparation of diaminodiacid building blocks

[0029] The reaction formula is:

[0030]

[0031] The specific steps are:

[0032] Compound 2: Take 1.5g (12.6mmol) of homoserine and 1.34g (12.6mmol) of sodium carbonate in a 150mL round-bottom flask, and dissolve them with 30mL of water and 15mL of 1,4-dioxane. The reaction system was stirred at 0°C, and 4.26 g (12.6 mmol) fluorenylmethoxycarbonyl succinimide (Fmoc-OSu) was added in batches. After the addition is complete, return the reaction system to room temperature (if there is undissolved solid in the reaction system, add an appropriate amount of 1,4-dioxane to dissolve it), and stir overnight. The 1,4-dioxane in the solvent was removed with a rotary evaporator, after which the remaining aqueous phase was extracted with ethyl acetate. Keep the aqueous phase and adjust the pH to 2 with 6M hydrochloric acid solution. The pH-adjusted aqueous phase was extracted with ethyl acetate, the organic phases were combined...

Embodiment 2

[0043] Preparation of Scorpion Toxin Polypeptide

[0044] The amino acid sequence of the scorpion toxin polypeptide is shown in SEQ ID No.1, and the S-S bond between the 8th and 28th positions is replaced by a C-S bond, which is equivalent to the S atom on Cys at the 28th position replaced by C atoms.

[0045] The reaction formula is:

[0046]

[0047] The specific steps are:

[0048] Weigh Rink AM amide Resin (0.1mmol) in a solid-phase synthesis tube, use DMF / DCM mixed solvent (1:1 volume ratio) to swell for 15 minutes, drain it, and then synthesize the above-mentioned Scorpion toxin polypeptide (0.4mmol amino acid, 0.4mmol Oxyma, 0.4mmol DIC; reaction time is 20-30min, reaction temperature is 75°C), in which arginine (Arg) uses HCTU as a condensation agent, and reacts twice at room temperature for 40min; Amino acid (His) uses DIC as the condensing agent, reacts at room temperature for 40 minutes; cysteine ​​(Cys) uses DIC as the condensing agent, reacts at 50°C for 40 ...

Embodiment 3

[0054] Activity Test of Scorpion Toxin Polypeptide

[0055] CHO cell culture and transfection process

[0056] Chinese hamster ovary (CHO) cells have no background current of potassium ion channels, and will not cause interference when studying potassium ion channels.

[0057] Cell culture medium: DMEM / F12 medium (Gibco Company) used for culturing cells. Add 10% fetal bovine serum (FBS) (Gibco) and 100 U / mL penicillin and streptomycin (Gibco) before use.

[0058] Cell Recovery: Turn on the 37 °C water bath. Prepare DMEM / F12 medium containing serum FBS and double-resistant penicillin and streptomycin, and place it in a 37°C water bath, preheating to 37°C. Take the cells out of the liquid nitrogen irrigation, put them into a water bath at 37 degrees Celsius quickly, put them into a centrifuge after they melt, centrifuge at 1000rpm for 5min, and discard the supernatant. The cells were resuspended with 1 mL of preheated DMEM / F12 medium, added to a 60 mm culture dish, and 2 mL ...

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Abstract

The invention discloses polypeptide of scorpion toxin. The amino acid sequence of the polypeptide of scorpion toxin is as shown in SEQ ID No.1, and the S-S bond between the site 8 and the site 28 is replaced by a C-S bond. The polypeptide of scorpion toxin disclosed by the invention has a quite stable structure. By introducing the C-S bond in synthesis of a peptide segment to replace the S-S bond which is easily broken, an action of stabilizing protein spatial conformation is played and pairing of residual disulfide bonds in a renaturation process is promoted to improve the integral renaturation efficiency. In addition, the method disclosed by the invention is simple to operate and high in yield. The obtained polypeptide of scorpion toxin is better in stability, relatively high in activity and nearly as same as natural scorpion toxin.

Description

technical field [0001] The invention relates to the field of protein polypeptide drugs, in particular to a scorpion toxin polypeptide and a synthesis method thereof. Background technique [0002] Natural scorpion toxin is a toxin protein found in scorpion venom and obtained through separation and purification. This protein has the potential drug potential for treating autoimmune diseases, so the toxin protein also has the potential of drug development. [0003] At present, the methods available to obtain scorpion toxin mainly include: [0004] One is directly extracted from the crude venom derived from scorpions, separated and purified by reverse-phase high-performance liquid chromatography C18 column, and various single components in the venom are separated from it. This method can obtain natural scorpion toxin polypeptide, but the disadvantage is that the amount of scorpion toxin obtained is very small. After 1 mg of crude poison is separated and purified, only micrograms...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/04C07K1/06A61P37/02
CPCC07K14/43522Y02P20/55
Inventor 田长麟张隆华郭晓奇陈晨晨肖亮周莉
Owner 合肥科生景肽生物科技有限公司
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