Recombinant Halase Expression in CHO Cells via Serum-Free Suspension Culture
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Solution Overview
Problem
The challenge lies in expressing recombinant Snake Venom Thrombin-Like Enzymes (SVTLEs) proteins, such as Haemocoagulase Acutus, which face issues with glycosylation modifications and stability in existing expression systems, affecting their physiological activity and scalability for industrial production.
Innovation Solution
The method involves optimizing the halase gene for mammalian cell expression using CHO cells in a serum-free suspension culture, constructing an expression vector, transfecting recombinant plasmids, and expressing the protein, which includes optimizing the gene sequence, designing primers for Not I and BamH I enzyme-cut sites, and using PEI for transfection to achieve stable and scalable production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a prokaryotic expression system (E. coli) is used to express SVTLEs recombinant protein, then the expression can be achieved, but the protein lacks proper glycosylation modification which greatly affects the activity of the protein
Solution Approach 1:
The patent changes the expression system from prokaryotic (E. coli) to mammalian (CHO cells) to alter the glycosylation capability parameter. This parameter change enables proper glycosylation modification of SVTLEs recombinant protein, thereby improving protein activity while maintaining industrial scalability through serum-free suspension culture technology.
2Productivity
If a yeast expression system is used to express SVTLEs recombinant protein, then the expression can be achieved, but the glycosylation modification is either excessive or lacks complex glycosyl chain modification which affects the functions of the recombinant protein
Solution Approach 1:
The patent changes the expression system from yeast to mammalian CHO cells to adjust the glycosylation parameter. CHO cells provide human-like glycosylation patterns with appropriate complexity and accuracy, resolving the issue of excessive or insufficient glycosylation modification while maintaining high expression efficiency through industrial-scale serum-free suspension culture.
3Reliability
If a traditional mammal cell expression system is used, then proper glycosylation can be achieved, but the system is easily infected by virus and has low automation level which limits industrial large-scale production
Solution Approach 1:
The patent changes the culture mode from traditional adherent culture to serum-free suspension culture in bioreactors. This parameter change enables automation and industrial large-scale production while maintaining proper glycosylation capability. The serum-free medium eliminates virus contamination risks associated with fetal bovine serum and allows for controlled, scalable production with high automation level.
4Quantity of substance
If snake venom is obtained from artificial feeding or wild catching, then the source is available, but there are huge differences in components and properties due to geographical and feeding conditions which restricts scale production and product homogeneity
Solution Approach 1:
The patent creates a recombinant copy of the SVTLEs protein in CHO cells that replicates the native protein's structure and function. This copying approach eliminates dependence on variable snake venom sources while maintaining the therapeutic activity. The recombinant system produces homogeneous, consistent batches suitable for industrial scale production, replacing the unstable natural source.
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
This approach successfully addresses the issues of raw material scarcity and quality instability, enabling efficient and stable production of recombinant halase proteins with preserved physiological activity, suitable for industrial-scale biopharmaceutical applications.
Implementation Method 1
expressing the recombinant protein and identifying
Implementation Method 2
expressing the recombinant protein and identifying
Implementation Method 3
using PEI for transfection to achieve stable and scalable production
Implementation Method 4
designing primers for Not I and BamH I enzyme-cut sites
Data Source
AI summary
The disclosure relates to an expression method of a Haemocoagulase Acutus (Halase) recombinant protein. The method includes the following steps: (1) optimizing a halase gene; (2) performing Polymerase Chain Reaction (PCR) amplification on an optimized halase gene; (3) constructing an Agkis-pMCX expression vector, transforming plasmids to competent cells of an Escherichia coli for amplification, screening in an Amp-resistant manner for positive cloning, sequencing and extracting recombinant plasmids with correct sequencing; (4) transfecting recombinant plasmids to CHO cells; and (5) expressing the recombinant protein and identifying. According to the expression method, a recombinant halase is expressed first using the CHO cells cultured in a serum-free suspension manner; and by utilizing a bioengineering means, the actual production problems of insufficient raw material sources and unstable quality of a snake venom product are solved successfully.