Synthetic method of polypeptide hydrazide containing cysteine residues and application of synthetic method

Abstract

The invention provides a synthetic method of polypeptide hydrazide containing cysteine residues and an application of the synthetic method. According to the synthetic method, a tetracysteine label isinserted into the C end of a target polypeptide, and a double-arsenic fluorescent dye labeling reagent is used for specific recognition and binding of the tetracysteine label sequence. Cysteine sulfydryl side chains in the target polypeptide are protected by Pac groups, then the double-arsenic fluorescent dye labeling reagent is removed, and cyano hydrazinolysis on the tetracysteine label sequenceis conducted to obtain the polypeptide hydrazide containing cysteine. The synthetic method is helpful for perfecting native chemical ligation reactions and more effectively synthesizing proteins.

Description

Technical field[0001]BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to the preparation of polypeptide / protein synthesis, and more particularly to synthesis methods of polypeptide acid hydrazide containing a cysteine ​​residue and its application.[0002]technical background[0003]The protein is an active substance in an organism that has the effect of constituent tissue, catalysis, regulation, and immune responses in the living body. It is the main responsibility of life activities. In order to clarify how proteins play a role in organisms, and modify the protein to obtain a wide concern of protein synthesis, protein synthesis is widely concerned.[0004]Natural Chemical Connections (NCL) is a reaction capable of efficiently connecting two-stage polypeptides. The reaction specifically procedure is the cysteine-based side chain of the N-terminal polypeptide to attack the carbonyl carbon of the C-terminal polypeptide sulfide, and the protein intermediates co...

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Problems solved by technology

The article (Angew.Chem.Int.Ed.2013,52:13062-13066) reported the sequence of Gly / His-Cys in 5% N 2 h 4 · HOAc, react at 60°C for 24 hours to obtain polypeptide hydrazide with C-terminal Gly / His, but this method is limited by the site and the reaction is not complete; article (ChemistryOpen.2015, 4, 448-452) It was reported that the peptides contained 10mM NiCl 2 , react in 50% methanol solution with pH 8.5 at 37°C for 3h, then add the final concentration of 5% NH 2 NH 2 ·H 2 O can obtain the polypeptide hydrazide with Ser as the cleavage site, but this method can only react under non-denaturing conditions, resulting in a decrease in the solubility of the polypeptide, and it is impossible to obtain the polypeptide hydrazide containing Ser; article (PeptideScience, 2015, 106, 531 -546) reported the cyanation of the Cys sulfhydryl side chain in the polypeptide, adding NH 2 NH 2 ·H 2 O hydrazinolysis can obtain the polypeptide hydrazide with Cys as the cleavage site, but this method cannot obtain the polypeptide hydrazide containing Cys. To solve this problem, this report uses Zn 2+ Selective protection of cysteine ​​through specific recognition and combination with zinc finger structure

The zinc finger sequence has more amino acid residues and realizes Zn 2+ Protection needs to be operated under non-denaturing conditions. However, most polypeptides / proteins are not highly soluble under non-denaturing conditions, so another method needs to be found to achieve selective protection of cysteine ​​and prepare the corresponding polypeptide hydrazide

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