1. Expression vector. From our previous transient expression experience, We noticed that a good expression vector for one protein is not necessary good for another protein. Empirically testing several different good expression vectors identify a best expression vector for a specific protein or antibody is necessary.
2. Signal peptide. Secreting signal is essential for targeting a protein to the secretary pathway. Several signal peptides, such as BSA, IgKappa, were most often used. Not all of the signal peptides are equal for a specific protein. We can screen several signal peptide and find the best one for your protein.
3. Transfection conditions. Lipids transfection is effective but could be very expensive when large amount of cultures need to be transfected. PEI or nanoparticles are less expensive and could possibly obtain the expression level comparable to lipids transfection reagents. Find the right way to do transfection could dramatically cut your cost.
4. Culture conditions. In general, antibodies are stable after secreted into the serum free medium. Longer incubation time usually increase the production level. However, some of the antibody could generate multimers and aggregates after prolonged culture. Therefore the culture time after transfection and culture condition including temperature is necessary to define.
Fig. Comparison of lipid reagents and PEI on HEK293 transfection.
- 1&2, Lipid Reagent 1: 2:1 and 4:1
- 3&4, Lipid reagent 2: 2:1 and 3:1
- 5&6, PEI 3:1 and 4:1
- 7&8&9, Lipid reagent 3: 0.85:1, 1:1, 1.4:1
- 10&11&12, 293fectin 1.5:1, 2:1, 3:1
Optimization for transient expression: Expression vector, signal peptide, transfection condition and culture condition
Optimization for stable pool: Expression vector, signal peptide, selection condition.