Recombinant Expression in Mammalian Cells
Mammalian cells are the primary option for the production of recombinant proteins and antibodies in pharmaceutical research. These cells are uniquely suitable to produce recombinant proteins with authentic mammalian PTM patterns and folding processes. In addition, endotoxin level is much lower for mammalian cell-produced proteins.
Bon-Opus offers mammalian cell expression service. Below is an outline for a typical mammalian expression project, and some key notes for each of the steps.
1) Designing Phase
1a) Determine the feasibility of mammalian expression:
Mammalian cells do have certain limitations. They are highly capable to drive expression of secreted proteins and extracellular domain of the proteins. However, it is very difficult to produce transmembrane and nuclear proteins from mammalian cells.
1b) Selection of proper signal peptides:
Based on the protein sequence, we will determine which signal peptide to use. The options include common signal sequence for secretion expression, such as IL2 lead sequence, and our in-house developed lead sequences.
1c) The use of epitope tags and position of the tag:
This will be decided by the application of the protein. You can find the pros and cons of the commonly used tags listed in the table below.
Once a protein of interest is identified, the AA will be subjected to codon optimization to increase translational efficiency. The optimized gene will be synthesized and sub-cloned into our in-house developed expression vector. The entire process can be completed within 3 weeks with the support of our molecular biology services.
2) Molecular Biology
3) Pilot Evaluation
Once an expression vector is made, a trial transfection will be conducted to determine the rough yield of the protein. The purified samples from the pilot can also be used to test its bioactivity in POC experiment.Once an expression vector is made, a trial transfection will be conducted to determine the rough yield of the protein. The purified samples from the pilot can also be used to test its bioactivity in POC experiment.
Based on your quantity need and the rough yield obtained from the pilot evaluation, a scale-up transfection will be carried out. This normally takes 2 weeks to complete.
4) Scale-up Production
5) Protein Purification
Based on the epitope tags and purity demand, affinity purification (protein A and his) along with other purification methods (IEX, gel-filtration, HIC, etc.) will be carried out to purify the target protein. In addition, endotoxin cleanup can also be performed upon request after purification is completed.
Biotinylation will be carried out after purification of the protein is completed. The degree of biotinylation will be measured by standard HABA assay. Avitag-based biotinylation will be done through BirA-mediated in vitro enzymatic reaction. NHS-based labeling will be performed using standard reagents. A labeling degree of 0.5 is guaranteed.
7) Final QC and Delivery
After all procedures are completed, the product will be subjected to a round of final QC, which may include SDS-PAGE and HPLC testing, as well as endotoxin tests. All the yield will be aliquoted and shipped to the customer as frozen liquid (dry ice shipment).