Recombinant Small-Molecule Collagen With Defined Sequence and High Yield
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Solution Overview
Problem
Existing recombinant small-molecule collagens face challenges in achieving a balanced molecular weight, maintaining biological activity, ensuring high expression levels, and achieving a single, defined amino acid sequence, particularly in eukaryotic expression systems like Pichia pastoris, with methods like tandem expression introducing non-essential amino acids and increasing purification costs.
Innovation Solution
A recombinant small-molecule collagen with a defined amino acid sequence and molecular weight, expressed using a tandem repeat expression strategy in Pichia pastoris, utilizing enzymes like Kex2 and CPB for intracellular cleavage, avoiding exogenous protein residues and enabling large-scale production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If enzymolysis or hydrolysis is used to produce small-molecule collagen peptides, then transdermal absorption is improved, but amino acid sequence control and molecular weight uniformity deteriorate
Solution Approach 1:
The collagen sequence is segmented into small monomer units (15-100 amino acids) that are then tandemly repeated to form the complete collagen structure. This segmentation allows each monomer to be precisely controlled while maintaining the overall collagen functionality and enabling transdermal absorption.
Solution Approach 2:
The molecular weight is precisely controlled by adjusting the number of tandem repeats and the length of individual monomers. By changing these parameters, the collagen can be optimized for both transdermal absorption (smaller size) and structural integrity (adequate size), achieving a balance that enzymolysis cannot provide.
2Reliability
If high molecular weight is maintained for biological activity, then biological activity is improved, but transdermal absorption deteriorates
Solution Approach 1:
The tandem repeat structure creates a continuous collagen-like sequence that maintains biological activity through repetition of functional motifs. This continuous structure ensures that even at smaller sizes, the collagen retains its ability to interact with cellular receptors and promote tissue regeneration.
Solution Approach 2:
By precisely controlling the molecular weight through defined repeat numbers and monomer lengths, the invention creates a size range that is small enough for transdermal absorption yet large enough to maintain biological activity, resolving the trade-off between these two critical properties.
3Productivity
If tandem expression is used to increase expression levels, then productivity is improved, but non-essential amino acid residues are introduced
Solution Approach 1:
The invention extracts and removes the non-essential amino acid residues (such as Glycine-X-Y repeats that are not part of the functional collagen sequence) through site-specific cleavage. This extraction allows high expression levels to be maintained while ensuring the final product contains only the essential collagen-determining amino acid sequences.
Solution Approach 2:
A site-specific cleavage site is introduced as an intermediary element that enables the removal of non-essential sequences. This cleavage site acts as a mediator between the tandem repeat expression system and the final pure collagen product, allowing high productivity while maintaining sequence purity.
4Manufacturing precision
If defined amino acid sequence is achieved through recombinant methods, then manufacturing precision is improved, but expression levels in eukaryotic systems deteriorate
Solution Approach 1:
The collagen sequence is divided into manageable monomer units that are tandemly repeated. This segmentation makes the recombinant expression more efficient in eukaryotic systems while maintaining precise amino acid sequence control, as each monomer can be independently optimized and the repetition amplifies expression levels.
Solution Approach 2:
By optimizing parameters such as monomer length (15-100 amino acids) and repeat number, the invention achieves both high expression levels and precise sequence definition. The tandem repeat structure is particularly amenable to recombinant expression, resolving the contradiction between precision and productivity.
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
The method achieves a 4-5 times yield increase, maintaining biological activity, and ensures a single, defined molecular weight, suitable for transdermal absorption and large-scale industrial production, with improved cell adhesion and proliferation-promoting activities.
Implementation Method 1
utilizing enzymes like Kex2 and CPB for intracellular cleavage
Data Source
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AI summary
The present invention provides a recombinant small-molecule collagen, an expression system thereof and a preparation method thereof, and belongs to the fields of synthetic biology, genetic engineering, and biotechnology. The present invention provides a recombinant small-molecule collagen derived from a plurality of sources of type-III and type-XVII collagens, and successfully expresses the recombinant small-molecule collagen. The present invention establishes one expression system of a small-molecule collagen and a preparation method of the small-molecule collagen, and includes an engineered exclusive chassis cell, a designed tandem repeat sequence of the small-molecule collagen, and a recombinant vector. The expression system or method of the present invention significantly enhances the yield of the small-molecule collagen. The present invention avoids the cost caused by in vitro protease digestion and the risk of exogenous protein residues, while also reducing the time and cost of a subsequent purification process.