Recombinant Human Type IV Collagen Production in E. coli for Scalable Purity
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Solution Overview
Problem
Existing methods for producing collagen, such as acid-hydrolysis, alkali-hydrolysis, or enzymatic-hydrolysis, result in collagen derivatives that lose biological activity, exhibit poor water solubility, and pose risks of viral infection and sensitization, while conventional recombinant expression methods are costly and not suitable for large-scale production.
Innovation Solution
A recombinant collagen is produced through large-scale screening and expression in Escherichia coli, utilizing specific amino acid sequences (SEQ ID NO: 1 or 28) or variants with high identity, linked via a linker, and purified using Ni column and strong anion exchange chromatography, suitable for high yields and cell adhesion activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional acid-hydrolysis, alkali-hydrolysis, or enzymatic-hydrolysis methods are used to produce collagen, then collagen derivatives can be extracted from animal tissues, but the collagen loses biological activity, exhibits poor water solubility, and poses risks of viral infection and sensitization
Solution Approach 1:
The patent uses recombinant DNA technology to create a synthetic copy of human type IV collagen in E. coli bacteria. The collagen gene is cloned into an expression vector and transformed into E. coli, which then produces recombinant human type IV collagen that mirrors the original human protein structure and biological activity, avoiding the need to extract from animal tissues
Solution Approach 2:
The patent introduces E. coli bacteria as an intermediary system to produce human collagen. The bacteria serve as a safe intermediary that can be fully controlled and sterilized, eliminating viral infection risks while producing biologically active human collagen through recombinant expression
2Reliability
If conventional recombinant expression methods are used to produce human collagen in vitro, then biological activity is maintained, but the production is costly and not suitable for large-scale manufacturing
Solution Approach 1:
The patent uses E. coli bacteria as a cheap, easily cultivable host system for collagen production. The bacteria can be grown rapidly in simple media at low cost, and the entire production system can be disposed of after use, eliminating the need for expensive mammalian cell culture facilities and reducing overall production costs
Solution Approach 2:
The patent optimizes several parameters to enable large-scale production: using a strong promoter (T7 promoter) for high-level expression, optimizing induction conditions (IPTG concentration, temperature, time), and scaling up from flask to fermenter culture. These parameter changes transform the system from research-scale to industrial-scale production
3Productivity
If recombinant collagen is produced through large-scale screening and expression in E. coli, then yields and purity are improved, but the production process becomes more complex
Solution Approach 1:
The patent divides the production process into distinct modular stages: (1) gene cloning and vector construction, (2) transformation into E. coli, (3) induction and expression, (4) cell harvesting and lysis, (5) purification via Ni-NTA chromatography, and (6) validation. This segmentation allows each step to be optimized independently and facilitates scale-up from laboratory to industrial production
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 achieves higher yields, purity, and cell adhesion activity compared to bovine type I collagen, suitable for various biomedical applications.
Implementation Method 1
purified using Ni column and strong anion exchange chromatography
Data Source
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AI summary
Provided herein is a method of biosynthesizing human body structural material type IV collagen. Provided herein is a recombinant collagen including the sequence represented by SEQ ID NO: 1, etc. The recombinant collagen of the present application exhibits excellent cell - adhesion activity. The method of the present application utilizes genetic engineering techniques to produce the recombinant collagen, thereby overcoming the drawbacks of the prior art.