Recombinant Type IV Collagen for Correct In Vitro Expression
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Solution Overview
Problem
Conventional methods struggle to correctly express and mass-produce human type IV collagen in vitro due to its complex structure and the challenges of forming a correct spatial structure without procollagen heads and tails, leading to loss of biological activity and limited application in biomedical fields.
Innovation Solution
A recombinant type IV humanized collagen is developed by screening functional domains of native human type IV collagen, using them as repeating sequences, and incorporating a TEV protease excisable sequence for expression and purification, facilitated by specific fermentation and chromatography processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional extraction methods (acidolysis, alkaline hydrolysis, enzymolysis) are used to obtain collagen, then collagen can be extracted from animal tissue, but the extracted collagen loses its original biological activity and cannot be applied to fulfill its true function in the biomedical field
Solution Approach 1:
The patent changes the fundamental parameter of collagen production from chemical extraction to biological synthesis. By using recombinant DNA technology to express human type IV collagen in eukaryotic expression systems, the collagen maintains its native structure and biological activity while being produced in sufficient quantities for biomedical applications.
Solution Approach 2:
The patent creates a copy of human type IV collagen through recombinant DNA technology. The human collagen gene is inserted into eukaryotic expression vectors, which then produce identical or highly similar collagen proteins that retain the original biological activity, avoiding the need for animal tissue extraction.
2Manufacturing precision
If the complete gene sequence of native human type IV collagen is used for expression, then the full-length collagen can be produced, but it cannot be spontaneously organized to form a correct spatial structure in vitro without procollagen heads and tails, leading to loss of biological activity
Solution Approach 1:
The patent segments the collagen protein into functional domains that can be independently expressed and assembled. By dividing the collagen sequence into specific repeating units and functional domains, the patent enables correct spatial organization in vitro while maintaining biological activity, avoiding the complexity of expressing the complete native gene sequence.
Solution Approach 2:
The patent performs preliminary design and optimization of the collagen sequence before expression. Functional domains are identified and engineered to facilitate spontaneous self-assembly into correct triple-helical structures in vitro, eliminating the need for procollagen heads and tails while ensuring proper spatial conformation.
3Quantity of substance
If animal-derived collagen is used, then collagen can be obtained from animal tissue, but the animal-derived immune response restricts its applications in the biomedical field
Solution Approach 1:
The patent creates a humanized copy of type IV collagen through recombinant DNA technology. By expressing human collagen sequences in eukaryotic systems, the patent produces collagen that is immunologically compatible with human patients, eliminating the immune response issues associated with animal-derived collagen while maintaining adequate supply for biomedical applications.
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 is correctly expressed in vitro, maintains biological activity, and achieves higher cell adherence, with a simpler and cost-effective production method.
Implementation Method 1
subjecting the recombinant host cell to fermentation culture, and inducing expression of a protein
Implementation Method 2
purifying the protein; preferably, comprising purifying the protein by a Ni affinity chromatography column and/or an ion exchange chromatography column
Data Source
AI summary
Disclosed in the present invention is a preparation method for a biosynthetic human body structural material. The present invention provides a recombinant humanized collagen type IV, the amino acid sequence thereof comprising n repetitive sequences, the n being an integer greater than or equal to 1, and the repetitive sequences being functional region sequences of native human collagen type IV The present invention provides the amino acid sequence of the recombinant humanized collagen type IV capable of being correctly expressed in vitro and biosynthesis thereof is successfully performed in vitro. The amino acid sequence of said protein is derived from the native human collagen type IV and does not cause adverse immune reactions when the protein is applied to a human body; in addition, compared with commercial humanized collagen, the recombinant humanized collagen type IV prepared by the present invention has a higher cell adhesion effect. Furthermore, the preparation method provided by the present invention is simple and can produce the recombinant humanized collagen type TV with a relatively high yield at a low cost.