Method for producing polypeptide fragment with high efficiency, which is suitable for NCL method

Abstract

The purpose of the invention is to provide a method for producing a polypeptide fragment with high efficiency, which is suitable for an NCL method. The production method comprises: a step of reacting a polypeptide which comprises a first polypeptide fragment having a cysteine residue as an N-terminal thereof and a second polypeptide fragment with CNBr to produce the first polypeptide fragment having a cysteine residue as an N-terminal thereof and a third polypeptide fragment, wherein the first polypeptide fragment and the second polypeptide fragment are linked to each other through an intervening sequence -Cys-W-(His)n-Z-Met-; and a step of reacting the third polypeptide fragment with a compound represented by formula (I) and a compound represented by formula (II) sequentially to produce the second polypeptide fragment having a modified C-terminal.

Description

technical field [0001] The present invention relates to efficient production methods of polypeptide fragments suitable for NCL method. Background technique [0002] For protein synthesis, various methods such as biosynthesis, chemical synthesis, and cell-free synthesis are known. In the biosynthetic method, cells such as Escherichia coli are used to introduce DNA encoding a protein to be synthesized into the cells and express the protein to obtain the protein. Chemical synthesis is the sequential bonding of amino acids by organic chemistry methods to synthesize target proteins. In addition, cell-free synthesis refers to synthesizing proteins in a cell-free manner using enzymes and the like present in various cells such as Escherichia coli. These methods can be appropriately selected or used in combination depending on the purpose of use, size, or properties to be added to the protein. [0003] In the middle part of the amino acid sequence, to synthesize a protein having s...

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Problems solved by technology

[0010] In addition, in biosynthesis, although the full-length protein can be normally expressed, in the case of a peptide fragment, it may be recognized as an error in the cell, so it may be decomposed, etc., and cannot be expressed normally.

Then, after the biosynthesis of the full-length protein, when the desired peptide fragment is excised as a thioester body by the above method, the long-chain polypeptide on the C-terminal side of the excised peptide fragment cannot be used.

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