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Method for preparing conotoxin polypeptide Eb1.6

A technology of eb1.6 and Cono snails, applied in the biological field, can solve the problems of unfavorable manual mass synthesis, inconvenience of condensation reaction and resin washing, and high cost, and achieves correct disulfide bond pairing method, high recovery rate, and realizes repeated recycling. Effect

Inactive Publication Date: 2013-01-16
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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] Embodiment 1, the large-scale preparation of cone snail polypeptide Eb1.6

[0034] 1. Exploration of the conditions for large-scale preparation of the cone snail polypeptide Eb1.6

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

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

Embodiment 2

[0066] Embodiment 2, the detection of polypeptide Eb1.6

[0067]The above-mentioned purified polypeptide Eb1.6 obtained in Example 1 was sequenced, and its amino acid sequence was sequence 1 in the sequence table, and then the polypeptide was further detected as follows:

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

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

[0070] First, the linear peptide Eb1.6 was synthesized (synthetic method is the same as step 1 of Experiment 2 in Example 1 above). The first step was air oxidation folding to form the first pair of disulfide bonds (Cys1-Cys3), and then the iodine was oxidized to remove the Acm protecting group to form the first pair of disulfide bonds. Two pairs of disulfide bonds (Cys1-Cys3, Cys2-Cys4), the first step of oxidative folding conditions: 0.1M Tris-HCl buffer, pH = 7.7, magnetic stirring for about 28h, HPLC analysis of the folding process. The fo...

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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 invention relates to the field of biotechnology, in particular to a method for preparing conotoxin polypeptide Eb1.6. Background technique [0002] Cono snails are gastropod molluscs. There are more than 500 species in the world, and they are found in warm sea areas all over the world. There are more than 100 species of cone snails in my country, mainly distributed in Xisha Islands, Hainan Island and Taiwan waters. Cono snail polypeptides are secreted by the venom glands of the cone snail venom tube and the inner wall of the poison sac. The venom of each cone snail contains 50-200 active peptides. Different species of cone snails contain different active peptides. Even the same species of taro Depending on the sea area of ​​the snail, the toxin composition may also be different. Theoretically, it is estimated that there are more than 50,000 polypeptides with different activities (McIntosh JM, Jones RM. Toxicon, 2001, 39: 1447-1451.). α-conopeptide...

Claims

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Application Information

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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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