Recombinant Type-I Humanized Collagen for Immune-Compatible Production
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Solution Overview
Problem
Existing collagen products face challenges such as low biological activity, immune response induction, high production cost, long production cycles, and limited applicability due to sourcing issues, making them unsuitable for biomedical use.
Innovation Solution
A recombinant type-I humanized collagen polypeptide with specific amino acid sequences (SEQ ID No: 3) is produced using a method involving expression in Escherichia coli, purification, and optional TEV protease cleavage, allowing for high yield and low cost production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional animal-derived collagen is used, then collagen products can be obtained, but they induce immune responses and have low biological activity
Solution Approach 1:
The patent creates a recombinant humanized collagen polypeptide that copies the essential functional sequences of natural human collagen (specifically the (Gly-Pro-Hyp)7 repeating unit) while using a heterologous expression system. This allows production of collagen with high biological activity similar to human collagen without using actual human tissue, thereby avoiding immune rejection and ethical issues.
Solution Approach 2:
The patent modifies the collagen sequence parameters by designing a humanized version with specific repeating units that maintain biological activity. The sequence is optimized for expression in E. coli while preserving the triple-helix structure and cell adhesion properties of natural collagen, achieving a balance between immunocompatibility and functionality.
2Quantity of substance
If traditional collagen extraction methods are used, then collagen can be obtained from animal tissues, but the production cost is high and the production cycle is long
Solution Approach 1:
The patent replaces traditional mechanical and chemical extraction methods (acid/alkaline treatment, enzymatic hydrolysis) with a biological expression system. By using recombinant DNA technology and E. coli expression, the patent achieves collagen production through cellular synthesis rather than tissue extraction, dramatically reducing production time and cost while scaling up efficiently.
Solution Approach 2:
The patent incorporates a TEV protease recognition sequence (ENLYFQ) at the N-terminus of the collagen polypeptide before expression. This allows for facile post-expression cleavage to remove the His-tag and obtain pure collagen, streamlining the purification process and reducing production time compared to traditional extraction methods that require multiple complex steps.
3Reliability
If traditional collagen extraction methods are used, then collagen can be obtained, but it loses original biological activity
Solution Approach 1:
The patent replaces harsh chemical extraction and enzymatic hydrolysis with gentle cellular expression and enzymatic cleavage. The recombinant expression system produces native-like collagen with preserved biological activity, while the TEV protease cleavage provides a mild, specific method for removing tags without damaging the collagen structure or activity.
Solution Approach 2:
The patent uses a fusion protein strategy where the collagen sequence is initially expressed with a His-tag and TEV protease recognition sequence. This intermediary construct allows for easy purification via nickel affinity chromatography followed by specific cleavage, providing a gentle manufacturing process that preserves biological activity unlike direct chemical extraction methods.
4Reliability
If human placenta is used as collagen source, then human collagen can be obtained, but the source is limited and legal restrictions apply
Solution Approach 1:
The patent copies the essential functional elements of human collagen sequences (the (Gly-Pro-Hyp)7 repeating unit characteristic of human type I collagen) and expresses them in a heterologous E. coli system. This eliminates dependence on limited human tissue sources like placenta while maintaining human collagen compatibility and avoiding legal and ethical restrictions.
Solution Approach 2:
The patent creates a universal production platform using E. coli expression that can produce humanized collagen without being constrained by the availability or legality of specific human tissue sources. The system is scalable, ethically acceptable, and legally compliant while maintaining the desired human collagen properties.
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 recombinant collagen exhibits enhanced cell adhesion properties and avoids immune responses, offering a cost-effective solution for biomedical applications.
Implementation Method 1
TEV Protease is a cysteine protease of Tobacco Etch Virus (TEV) with His tag (6×His tag) recombinantly expressed in Escherichia coli, and it can specifically recognize heptapeptide sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser and perform enzymatic cleavage between Gln and Gly/Ser amino acid residues
Implementation Method 2
the repetitive sequences are directly linked to each other when n is an integer greater than or equal to 2; optionally, the N-terminus of the recombinant type-I humanized collagen polypeptide comprises an amino acid sequence capable of being cleaved off by TEV protease
Data Source
AI summary
Disclosed in the present invention are a recombinant type-I humanized collagen polypeptide, and a preparation method therefor and the use thereof. The recombinant type-I humanized collagen polypeptide provided by the present disclosure comprises n repeats of a sequence as shown in SEQ ID NO: 1, wherein n is an integer greater than or equal to 1; the repetitive sequences are directly linked when n is an integer greater than or equal to 2; and optionally, the N-terminus of the recombinant type-I humanized collagen polypeptide contains an amino acid sequence capable of being cleaved off by the TEV protease. The recombinant type-I humanized collagen polypeptide provided by the present disclosure has an activity of promoting cell adhesion, the recombinant protein has an amino acid sequence selected from the amino acid sequence of natural collagen and does not generate immune response when applied to the human body, the preparation method therefor is simple, and high-yield collagen can be obtained at a low cost.