Recombinant Peptide Production Using Self-Assembling Precursor Proteins
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Solution Overview
Problem
Current methods for producing peptides using repetitive precursor proteins are not efficient, as they often result in low peptide proportions and require re-establishment of expression conditions for different sequences, and previous methods are not suitable for all peptide sequences.
Innovation Solution
A novel approach involving recombinant production of repetitive precursor proteins with a high proportion of desired peptide sequences and auxiliary sequences that dominate the protein's properties predictably, allowing for the production of different peptide sequences without altering expression conditions, using a precursor protein with a cleavable repetitive sequence of repeat units containing desired and auxiliary peptide elements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If fusion proteins are used for peptide production, then peptide stability is improved, but peptide proportion in the precursor protein becomes low
Solution Approach 1:
The precursor protein is divided into multiple repeat units, each containing a peptide sequence flanked by cleavage sequences. This segmentation allows the peptide to constitute a significant portion of the precursor protein while maintaining stability through the fusion protein approach.
2Productivity
If repetitive precursor proteins are used to increase peptide proportion, then peptide yield is improved, but expression conditions must be re-established for different sequences
Solution Approach 1:
The auxiliary sequence is designed to provide universal stability and solubility properties that work across different peptide sequences. The standardized structure with N-terminal and C-terminal auxiliary sequences flanking repetitive peptide units creates a universal platform that can express various peptide sequences without requiring re-optimization of expression conditions.
3Reliability
If cationic antimicrobial peptide sequences are used, then desired peptide activity is achieved, but host cell damage occurs
Solution Approach 1:
An anionic auxiliary sequence acts as an intermediary between the cationic antimicrobial peptide and the host cell. This auxiliary sequence with negative charge (rich in glutamate and aspartate residues) neutralizes the positive charge of the antimicrobial peptide, preventing host cell damage while allowing the peptide to retain its antimicrobial activity against target organisms.
4Stability of the object's composition
If auxiliary sequences are added to protect host cells, then host cell stability is improved, but precursor protein complexity increases
Solution Approach 1:
The auxiliary sequence parameters are optimized to provide protection with minimal complexity. Specifically, the auxiliary sequence contains 15-30 residues with a net negative charge of -3 to -8 at pH 7, rich in glutamate and aspartate residues, forming an amphipathic helix structure. These defined parameters provide host cell protection while maintaining a relatively simple and predictable precursor protein structure.
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 production of peptides with high peptide proportions and predictable properties, suitable for various sequences, improving yield and simplifying processing steps.
Implementation Method 1
the precursor protein shows self-assembling properties, so that the precursor protein forms stable associates
Implementation Method 2
the peptidic link is chemically specifically cleavable
Data Source
Figure 1A~1B
Figure 2
Figure 3
AI summary
The present invention relates to repetitive self-assembling precursor proteins, nucleic acid sequences and expression constructs encoding the same, and to methods for recombinant production of peptides using such precursor proteins.