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Method for preparing conotoxin polypeptide Eb1.6

A technology of eb1.6 and cone snails, applied in the biological field, can solve the problems of unfavorable manual mass synthesis, condensation reaction, inconvenient resin washing, high cost, etc., achieve correct disulfide bond pairing mode, high recovery rate, and realize repeated recycling Effect

Inactive Publication Date: 2014-07-09
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is not conducive to manual large-scale synthesis, because the condensing agent DCC reacts to form N,N'-dicyclohexylurea (DCU) which is insoluble in N,N-dimethylformamide (DMF) or dichloromethane (DCM) , which brings inconvenience to the condensation reaction and resin washing
In addition, the Eb1.6 linear peptide is folded into the target substance using C18 column reversed-phase adsorption, acetonitrile elution, high cost

Method used

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  • Method for preparing conotoxin polypeptide Eb1.6
  • Method for preparing conotoxin polypeptide Eb1.6
  • Method for preparing conotoxin polypeptide Eb1.6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Scale preparation of Cono polypeptide Eb1.6

[0034] 1. The conditions for the large-scale preparation of Cono peptide Eb1.6

[0035] 1. The influence of different condensing agents on the efficiency of manual solid phase synthesis of Eb1.6 linear peptide

[0036] The pre-synthesis of Eb1.6 linear peptide was synthesized by an instrument, which was carried out on the 433A peptide synthesizer (ABI American Applied System Biology Company instrument), using Fmoc-protected amino acids (Shanghai Gill Biochemical Co., Ltd.) and a substitution rate of 0.60mmol / g Rink resin, condensation agent is DCC / HOBt. In the reaction system, the molar ratio of resin to amino acid is 1:5, and 0.1mmol peptide resin is synthesized each time, and the coupling rate is high ( figure 1 -A). However, this method is not conducive to manual large-scale synthesis, because the condensation agent DCC reacts to form N,N'-dicyclohexylurea (DCU) which is insoluble in N,N-dimethylformamide (DMF) or d...

Embodiment 2

[0066] Example 2. Detection of polypeptide Eb1.6

[0067] The purified polypeptide Eb1.6 obtained in Example 1 was sequenced, and the amino acid sequence was sequence 1 in the sequence table, and the polypeptide was further tested as follows:

[0068] 1. Determination of peptide Eb1.6 disulfide bond

[0069] The determination of the peptide Eb1.6 disulfide bond adopts a two-step folding method:

[0070] First, synthesize linear peptide Eb1.6 (synthesis method is the same as step 1 of experiment 2 in Example 1 above). In the first step, air oxidation folds to form the first pair of disulfide bonds (Cys1-Cys3), and then iodine is oxidized to remove the Acm protecting group to form the first Two pairs of disulfide bonds (Cys1-Cys3, Cys2-Cys4), the first oxidative folding conditions are: 0.1M Tris-HCl buffer, pH=7.7, magnetic stirring for about 28h, HPLC analysis of folding progress. After the folding is sufficient, the reaction is terminated with dilute acetic acid, and after the enrich...

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Abstract

The invention relates to a method for preparing conotoxin polypeptide Eb1.6. The method comprises the following steps of: 1) synthesizing Eb1.6 linear peptide by using a condensing agent N,N'-diisopropylcarbodiimide / 1-hydroxybenzotriazole (DIC / HOBt); and 2) folding the linear peptide to obtain the conotoxin polypeptide Eb1.6. According to the method, the efficiency of the manual synthesis of the linear peptide is improved by using the condensing agent DIC / HOBT; an MNH4HCO3 buffer solution with the pH value of 8.0 and the concentration of 0.1 is selected as a folding buffer solution, is cheap and readily available, and is high in folding rate; a product obtained by the folding is adsorbed by using AmberliteXAD16 macroporous adsorption resin, the resin is dipped by using ethanol, a dipping solution is concentrated, and then an obtained product can be directly used for the subsequent C18 column purification; and meanwhile, the resin washed by using the ethanol is washed by using water and is regenerated, so that the recycling of the buffer system and the resin can be realized, the recovery rate is relatively high, and the cost is reduced.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for preparing the conotoxin polypeptide Eb1.6. Background technique [0002] Cono is a gastropod mollusk. There are more than 500 species in the world, all over the warm sea areas of the world. There are more than 100 types of cono, mainly distributed in the Paracel Islands, Hainan Island and Taiwan waters. Cono snail polypeptides are secreted by the venom tube and venom glands of the inner wall of the venom sac. The venom of each type of Cono contains 50-200 active peptides. Different varieties of Cono contain different active peptides, even if they are the same species. Due to different sea areas, snails may have different toxin components. It is theoretically estimated that there are more than 50,000 polypeptides with different activities (McIntosh JM, Jones RM. Toxicon, 2001, 39: 1447-1451.). α-Cono polypeptide (α-CTX) belongs to the Cono polypeptide A superfamily, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/06C07K1/04
Inventor 戴秋云徐艳董铭心余硕刘珠果王孝花周尚民李海涛代琴
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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