Recombinant Collagen Expression in Expi293F Cells
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing collagen, such as using animal-derived sources or expression in Escherichia coli, face challenges like immunogenicity, poor uniformity, and difficulties in achieving full-length, soluble collagen with appropriate post-translational modifications.
Innovation Solution
A recombinant collagen is developed by combining amino acid sequences of type III human collagen and the α2-chain of type I human collagen, expressed using a human embryonic kidney cell line (Expi293F), and enhanced with secretion, histidine, fusion, and protease cleavage tags to improve expression and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If animal-derived collagen is extracted by acid method, alkali method, or enzymatic method, then complete triple-helical structure is obtained with low cost, but immunogenicity and poor uniformity occur
Solution Approach 1:
The patent uses recombinant DNA technology to copy the collagen gene into a host cell system, allowing production of collagen with identical amino acid sequences to human collagen. This genetic copying approach eliminates immunogenicity issues while maintaining the complete triple-helical structure and biological activity, resolving the contradiction between cost-effectiveness and immunogenicity.
Solution Approach 2:
The patent changes the production parameters from traditional animal-derived extraction methods to recombinant DNA expression systems. By altering the source material from animal tissues to genetically engineered cells, the patent achieves uniform human-like collagen structure while maintaining manufacturing efficiency, addressing both cost and reliability concerns.
2Ease of manufacture
If Escherichia coli is used to express collagen, then production cost is reduced, but soluble collagen expression is difficult and inclusion bodies form
Solution Approach 1:
The patent introduces a eukaryotic expression system (such as Chinese hamster ovary cells or human embryonic kidney cells) as an intermediary between the collagen gene and the final protein product. This intermediary system provides the necessary cellular machinery for proper collagen folding, solubility, and post-translational modifications, avoiding inclusion body formation while maintaining cost-effectiveness through scalable cell culture production.
Solution Approach 2:
The patent changes the expression system from prokaryotic (E. coli) to eukaryotic cell systems, fundamentally altering the cellular environment parameters. This parameter change enables proper collagen triple-helical structure formation, solubility, and secretion while maintaining economic viability through automated cell culture processes, resolving the contradiction between cost and manufacturing precision.
3Ease of manufacture
If full-length human collagen is expressed in yeast system, then production is simplified, but post-translational modifications differ from human body
Solution Approach 1:
The patent changes the expression system parameters from yeast to human or primate cell lines, matching the cellular environment required for authentic human collagen post-translational modifications. This parameter change ensures proper glycosylation, hydroxylation, and triple-helical structure formation while maintaining production simplicity through established cell culture techniques, resolving the contradiction between manufacturing simplicity and modification precision.
4Productivity
If recombinant collagen is produced in human embryonic kidney cell Expi293F, then expression level is significantly increased, but production complexity increases
Solution Approach 1:
The patent uses a standardized recombinant DNA expression platform in Expi293F cells, copying successful expression strategies from established biotechnology protocols. This standardized approach achieves high expression levels while managing complexity through modular vector design and automated cell culture processes, making the high productivity achievable without proportionally increasing operational complexity.
Data Source
AI summary
Provided are a recombinant collagen, an expression method and use thereof. In the present disclosure, the recombinant collagen is expressed by a human embryonic kidney cell Expi293F at a high protein expression level, thereby realizing the expression of a large-molecular-weight recombinant collagen. Activity studies have showed that the expressed recombinant collagen has desirable activity in promoting cell migration, while the recombinant collagen III+I with a fusion tag shows high stability at 4° C. In addition, cell activity studies have shown that the recombinant type III collagen expressed by human cells has better activity in promoting the cell migration than that of a commercially available type III collagen. These results indicate that the human cell expression system is conducive to the expression of highly active and large-molecular-weight recombinant collagen.