Chemical Synthesis of Large Mirror-Image Proteins via Segmentation
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Solution Overview
Problem
Current methods are limited in synthesizing large mirror-image proteins with more than 400 amino acid residues due to challenges in synthesis and ligation efficiencies of peptide segments, restricting the development of mirror-image biology systems and their applications.
Innovation Solution
The method involves identifying ligation-conducive sequences in the protein sequence, parsing them into segments, and chemically synthesizing these segments, with mutations introduced to reduce hydrophobicity and cost by substituting Ile residues, allowing for the chemical synthesis of large proteins through split protein design and native chemical ligation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If chemical synthesis methods are used to produce large mirror-image proteins, then the ability to create proteins with over 400 amino acid residues is enabled, but synthesis and ligation efficiencies decrease
Solution Approach 1:
The patent applies segmentation by dividing the large mirror-image protein into multiple smaller peptide segments that can be chemically synthesized separately with higher efficiency, then ligating them together to form the complete protein. This resolves the contradiction by enabling long protein synthesis while maintaining practical synthesis rates through parallel processing of smaller units.
Solution Approach 2:
The patent employs preliminary action by pre-synthesizing and purifying individual peptide segments before final assembly. This allows optimization of each synthesis step independently, ensuring high efficiency for each segment while achieving the overall goal of producing large proteins through systematic preparation and ligation.
2Ease of manufacture
If mutations are introduced to reduce hydrophobicity by substituting Ile residues, then solubility and synthesis feasibility improve, but protein sequence fidelity decreases
Solution Approach 1:
The patent applies local quality by introducing mutations specifically at ligation sites and hydrophobic regions where they facilitate synthesis and solubility, while maintaining the wild-type sequence in functional domains. This localized approach improves manufacturability without compromising the functional integrity and sequence fidelity of critical protein regions.
Solution Approach 2:
The patent uses parameter changes by systematically substituting hydrophobic residues (particularly Ile) with less hydrophobic alternatives at specific positions to improve solubility and synthesis feasibility. These controlled parameter modifications enhance manufacturability while preserving overall protein function through strategic rather than comprehensive sequence alteration.
3Reliability
If D-amino acids are used to create mirror-image proteins, then resistance to proteolysis and orthogonal biological tools are achieved, but production cost increases
Solution Approach 1:
The patent applies this principle by using chemically synthesized peptide segments with D-amino acids for specific applications where protease resistance is critical (such as mirror-image phage display and structure determination), rather than producing entire large proteins with D-amino acids. This selective use reduces costs by employing expensive D-peptide technology only where necessary while maintaining functional benefits.
Solution Approach 2:
The patent uses segmentation to divide the protein into D-peptide segments for protease-resistant regions and L-peptide segments for other regions, or synthesizes only critical functional segments as D-peptides. This approach achieves protease resistance in essential areas while reducing overall production costs by limiting the use of expensive D-amino acid synthesis to necessary portions rather than the entire protein.
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 approach enables the chemical synthesis of proteins with over 400 amino acids, maintaining functional activity and reducing production costs, facilitating applications in bio-orthogonal molecular data storage and other biological research.
Implementation Method 1
a conjunction between solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL) has yielded a powerful method that enabled the total chemical synthesis of various proteins
Data Source
AI summary
Provided herein is a general method for producing large (more than 400 aa long) D-amino acids proteins, also referred to as mirror image protein (with respect to their naturally occurring L-amino acids counterparts), including RNA/DNA manipulating enzymes, and uses thereof in a wide range of research, practical data storage and medicinal applications.