Polypeptide Fragment Synthesis for Native Chemical Ligation
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Solution Overview
Problem
Current methods for synthesizing peptide thioesters for the Native Chemical Ligation (NCL) method are limited by the size restrictions of solid phase synthesis and require complex procedures, making it difficult to obtain suitable polypeptide fragments for protein synthesis, especially for long chains and those with modifications like sugar chains.
Innovation Solution
A method involving the preparation of polypeptide fragments with specific sequences, including a cysteine at the N-terminal and a modified C-terminal, using CNBr treatment and subsequent reactions with specific compounds to obtain fragments suitable for NCL, allowing for efficient ligation and synthesis of modified peptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If solid phase synthesis method is used to prepare peptide thioester for NCL, then the peptide can be synthesized with defined sequence, but the peptide chain length is restricted to approximately 50 residues at the longest
Solution Approach 1:
The invention divides the long peptide chain into two separate peptide fragments that are synthesized independently and then ligated together through NCL. The first fragment contains the N-terminal portion and the second fragment contains the C-terminal portion, allowing each fragment to be synthesized within the capability limits of solid phase synthesis while the final ligation produces the full-length peptide exceeding 50 residues
Solution Approach 2:
The invention performs preliminary biosynthesis of the peptide fragments using an intein system before the final NCL step. The intein-mediated protein splicing is carried out in advance to generate the peptide fragments with appropriate termini, preparing them for the subsequent chemical ligation step
2Manufacturing precision
If NCL method is used to link peptide chains, then natural amide bond can be formed at ligation site, but complex procedures are required including preparation of peptide thioester form
Solution Approach 1:
The invention utilizes the self-processing capability of the intein system to automatically generate the required peptide thioester intermediate and facilitate the ligation reaction. The intein-mediated protein splicing system performs the complex chemical transformations autonomously in the biosynthetic system, eliminating the need for manual preparation of thioester forms and reducing overall procedure complexity
3Ease of manufacture
If peptide fragment is expressed as fusion protein with intein, then thioester form can be obtained, but the peptide sequence must allow functioning of protein splicing and proper folding
Solution Approach 1:
The invention extracts the peptide fragment of interest from the intein fusion protein through intein-mediated protein splicing. The intein system is used as a temporary vehicle for expression and processing, and the desired peptide fragment is released in the required thioester form, separating the function of thioester generation from the final peptide sequence requirements
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the efficient manufacture of polypeptide fragments suitable for NCL, facilitating the synthesis of long chain peptides with modifications, such as sugar chains, by combining biosynthesis and chemical synthesis, and allows for site-specific glycosylation, improving the overall protein synthesis process.
Implementation Method 1
reacting a polypeptide having the following structure: (N-terminal) second polypeptide fragment-Cys-W-(His)n-Z-Met-first polypeptide fragment (C-terminal) with CNBr to obtain the following polypeptide fragments
Implementation Method 2
the thiol group on the side chain of the cysteine (SH group, may be referred to as a sulfhydryl group) selectively reacts with the carbonyl carbon of the thioester group and a thioester bond early intermediate is produced by a thiol exchange reaction
Implementation Method 3
This intermediate is spontaneously intramolecularly rearranged, rendering a natural amide bond to the linking site, while regenerating the cysteine side chain thiol
Data Source
Figure 1
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Figure 5a~6
AI summary
[Problem] The object of the present invention is to provide a method for efficiently manufacturing a polypeptide fragment suitable for the NCL method. [Solution] The present invention provides a manufacturing method comprising a step of reacting a polypeptide consisting of a first polypeptide fragment having cysteine at the N-terminal and a second polypeptide fragment linked via an intervening sequence -Cys-W-(His)n-Z-Met- with CNBr to obtain a first polypeptide fragment having cysteine at the N-terminal and a third polypeptide fragment, and a step of sequentially reacting the third polypeptide fragment with a compound represented by the following formula (I) and a compound represented by the following formula (II) to obtain a second polypeptide fragment having the C-terminal modified.