Protein preparation method

A protein and hydrazide technology, applied in the field of peptide connection, can solve the problems of inefficient synthesis, unstable HF, and high corrosiveness

Active Publication Date: 2011-09-28
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The thioesterification of polypeptide needs to be synthesized by Boc-SPPS method, but Boc-SPPS needs to use highly corrosive HF
In addition, glycosylated, phosphorylated and sulfated peptides are unstable to HF and cannot be synthesized efficiently
Therefore, the current protein synthesis method needs to be improved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, synthetic hydrazide resin

[0076] 1. Swell Wang resin (1.5 g) with DCM (14 mL), put it into a round-bottomed flask, add N-methylmorpholine (0.16 mL) and p-nitrophenoxychloride in turn in an ice-salt bath with stirring (0.29 g), after the system was stirred overnight at room temperature, the resin was transferred to a sand core funnel, the resin was washed with a large amount of DCM / DMF / MeOH / DCM, and the resin was dried overnight with a vacuum dryer.

[0077] 2. Put the Wang resin of p-nitrophenoxycarbonylation (purchased from Tianjin Nankai Hecheng Technology Co., Ltd.) into a round bottom flask, add the configured precooled mixed solution (composition: hydrazine hydrate: DMF: DCM = 0.16mL: 30mL: 22mL), stirred overnight at room temperature. The resin was transferred to a sand core funnel, the resin was washed with plenty of DCM / DMF / MeOH / DCM, and the resin was dried overnight with a vacuum desiccator. That is, the hydrazide resin used for synthesizing p...

Embodiment 2

[0078] Embodiment 2, Fmoc method solid-phase synthesis polypeptide hydrazide

[0079] 1. Add the hydrazide resin to the solid phase reactor and swell it with 1:1 DMF / DCM for 3 hours, and now configure the mixed solution (3.6 equivalents of HBTU: 4 equivalents of HOBt: 8 equivalents of DIEA: 0.1 equivalents of DMAP: 4 equivalents of the protected target peptide The first amino acid at the C-terminal) was added to the resin to react for 8 hours, and the resin was washed successively with a large amount of DMF, DCM, and DMF. After the masking reagent (acetic anhydride: DIEA: DMF = 1:1:8) soaked the resin for 10 minutes, the resin was washed again with a large amount of DMF, DCM, and DMF. Add 20% piperidine in DMF to soak the resin for 5 minutes and 10 minutes. After washing with a large amount of DMF, DCM, and DMF, add the currently prepared mixed solution (3.6 equivalents of HBTU: 4 equivalents of HOBt: 8 equivalents of DIEA: 4 equivalents of the second amino acid at the C-term...

Embodiment 3

[0081] Example 3, synthesis of H-Leu-Tyr-Arg-Ala-Tyr-Cys-Lys-Tyr-Met-His-OH by polypeptide hydrazide method

[0082] 1. Weigh the polypeptide hydrazide H-Leu-Tyr-Arg-Ala-Tyr-NHNH 2 (1.5mg), H-Cys-Lys-Tyr-Met-His-OH (1.95mg) dissolved in 0.65mL PBS (6.0M guanidine hydrochloride, 0.2M phosphate, pH 3.0), plus 50uL benzamide solution (40mM) .

[0083] 2. Take 0.25mL of the above solution and put it into a 1.5mL glass bottle, add a magnet, and add 25uL NaNO dropwise under stirring in an ice-salt bath (about -10°C) 2 (0.2M) aqueous solution, react at low temperature for about 20 minutes.

[0084] 3. After oxidation, add 0.25mL MPAA guanidine hydrochloride solution (0.2MMPAA, 6.0M guanidine hydrochloride, 0.2M phosphate, pH 5.0) dropwise, remove the ice-salt bath, take NaOH (2.0M) solution to carefully adjust the acidity of the reaction solution to neutral (acidity measured with a micro glass pH meter).

[0085] 4. After 2 hours of reaction, take 20uL of the reaction solution an...

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Abstract

A protein preparation method provided by the invention comprises the following steps to connect a first peptide with hydrazide group on C terminus and a second peptide with cysteine on N terminus: mixing the first peptide and the second peptide in a solution containing NaNO2 and adjusting the mixed solution pH to an acidic pH so as to converting the hydrazide group of the first peptide into acyl azide group to obtain a first reaction mixture; adding a solution containing a reducing agent into the first reaction mixture and adjusting the pH to a neutral pH, wherein the reducing agent contains mercapto group, so as to convert the acyl azide group of the first peptide into thioester group, and maintaining the neutral pH so as to conducting a connection reaction between the thioester on the Cterminus of the first peptide and the N terminus of the second peptide to produce proteins. A method for preparing polypeptide thioester compounds is also provided. According to the protein preparation method provided by the invention, proteins can be effectively synthesized.

Description

technical field [0001] The present invention relates to the preparation of proteins and methods of proteins, more particularly to methods of linking peptides. Background technique [0002] The relationship between protein structure and function is one of the frontier hot issues in biology, and the acquisition of high-purity or specific non-naturally modified proteins can greatly promote the development of this field. Chemical synthesis and semi-synthesis of proteins are considered to be important ways to obtain target proteins with specific structural modifications, so the development of efficient synthesis techniques is of great academic and commercial value. As early as 1901, Fisher, a master of organic synthesis, proposed the idea of ​​chemically synthesizing biological enzymes. In 1963, Merrifield developed solid-phase synthetic peptide technology to realize the chemical synthesis of biologically active peptides. However, due to the serious aggregation of long-chain po...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/107
Inventor 刘磊方葛敏李宜明沈非黄轶超李佳斌林云崔洪奎
Owner TSINGHUA UNIV
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