简介：
Overview
This protocol outlines a quick and cost-efficient method for producing secreted, glycosylated mammalian proteins. It emphasizes the importance of cell health and monitoring conditions to achieve high yields suitable for structural studies.
Key Study Components
Area of Science
- Protein Expression
- Biophysical Studies
- X-ray Crystallography
Background
- Focus on producing natively folded proteins.
- Importance of cell viability and media conditions.
- Monitoring cell density is crucial for yield.
- Utilizes 293F cells for large scale culture.
Purpose of Study
- To develop a fast protocol for obtaining mammalian proteins.
- To ensure proteins are suitable for structural studies.
- To improve protein yield through careful monitoring.
Methods Used
- Transfection of 293F cells.
- Supplementation of culture media with glutamine and penicillin-streptomycin.
- Monitoring of cell health and media sugar levels.
- Large scale culture techniques.
Main Results
- Achieved milligram quantities of secreted proteins.
- Identified optimal conditions for cell growth and protein yield.
- Demonstrated the effectiveness of monitoring cell density.
- Provided a reliable method for protein purification.
Conclusions
- This protocol is efficient for producing high-quality mammalian proteins.
- Cell health and monitoring are key to successful protein expression.
- Suitable for various structural and biophysical applications.
What are the main advantages of this protocol?
The protocol is fast, straightforward, and yields high-quality proteins suitable for structural studies.
How does cell density affect protein yield?
Exceeding 2 million cells per milliliter before transfection can significantly reduce protein yield.
What media supplements are recommended?
It is recommended to supplement 1 liter of 293F media with 10 mL of 100x glutamine and 5 mL of 100x pen-strep.
Why is monitoring cell viability important?
Monitoring cell viability helps to maximize protein yield and prolongs the usefulness of the cells.
What type of proteins does this protocol produce?
The protocol produces secreted, glycosylated mammalian proteins that are natively folded.
Can this method be used for large-scale protein production?
Yes, this method is designed for large-scale culture of 293F cells.
繁體中文
