In transient expression, various parameters can be optimized to enhance protein yield, transfection efficiency, and overall expression performance. Key parameters include: 

1.  DNA Quality and Quantity: 

     ●      Optimization: Use high-quality, endotoxin-free plasmid DNA. The amount of DNA used per transfection can also be optimized to balance between cytotoxicity and transfection efficiency. 

     ●      Impact: Improves transfection efficiency and protein expression levels. 

2.  Transfection Reagent: 

     ●      Optimization: The type and ratio of transfection reagent to DNA are critical. Different reagents (lipid-based, polymer-based, or electroporation) can be tested to find the best fit for the specific cell type. 

     ●      Impact: Affects the efficiency of DNA uptake by cells, directly influencing expression levels. 

3.  Cell Density: 

     ●      Optimization: Cells should be in optimal growth phase, usually at 60-80% confluency at the time of transfection. 

     ●      Impact: Proper cell density ensures healthy cells and high transfection efficiency, leading to better protein expression. 

4.  Culture Medium: 

     ●      Optimization: Some cell lines benefit from the use of serum-free or specialized media. Additionally, medium additives such as sodium butyrate can enhance expression levels. 

     ●      Impact: Ensures proper cell health and boosts protein production. 

5.  Transfection Time: 

     ●      Optimization: The time between transfection and protein harvest can be adjusted. Protein expression typically peaks 24-72 hours post-transfection, depending on the cell type and vector. 

     ●      Impact: Determines when the maximum protein yield can be harvested.

6.  Plasmid Design: 

     ●      Optimization: Optimizing the plasmid vector for efficient transcription and translation, including strong promoters (e.g., CMV, SV40), codon optimization, and adding enhancer elements. 

     ●      Impact: Ensures robust expression of the target gene. 

7.  Temperature Post-Transfection: 

     ●      Optimization: Lowering the temperature (e.g., from 37°C to 30°C) after transfection can increase protein folding and stability. 

     ●      Impact: Helps improve the quality of the expressed protein, especially for secreted or complex proteins. 

The main parameters for optimizing transient expression include DNA quality and quantity, transfection reagents, cell density, culture medium, transfection time, plasmid design, and post-transfection temperature. Fine-tuning these factors can significantly improve transfection efficiency, protein yield, and expression quality. 

GenCefe provide one-stop solutions from codon optimization, gene synthesis, to protein expression and purification. In addition to four conventional expression systems of bacterial,yeast,insect cell, and mammalian cell, we have also developed a patented cell free expression technology, which is suitable for the production of difficult proteins such as toxic proteins and membrane proteins.