Mammalian cells are the preferred expression platform for proteins requiring fully mammalian post-translational modifications. As such, they are commonly used for the production of antibodies and therapeutic proteins. They are also preferred when the
downstream application for a recombinant protein is in a functional mammalian cell-based assay. CTSR typical time line is 2-4 weeks from DNA delivery to harvesting, add another 1-2 weeks for purification.
- Phase I: Gene Synthesis and Subcloning – performed by Research Lab
- Customer produces cDNA of any desired coding sequence and subclone it into a mammalian expression vector. Once cloned, all constructs are restriction digest and sequence verified. Customer provides required amount of DNA for size of
transfection (general-3 μg DNA/ml transfection vol).CTSR DNA calculation is 1.5ug of DNA/ml of transfection, example 500ml flask requires 750ug of purified STERILE DNA.
- Phase II: Pilot Protein Expression
- Generally, we conduct small scale expression trials to identify the best clones and/or expression conditions. We use these conditions to continue on the Phase III.
- Phase III: Protein Expression
- For larger protein requests, we scale up expression using optimized conditions. We utilize 2-10L Wave Bag systems to produce gram quantities of the desired protein.
The baculovirus expression vector system (BEVS) is a powerful system for the production of high quality proteins. Although time lines are increased compared to E. coli, proteins produced in BEVS tend to be soluble and functional and carry post-translational
modifications like phosphorylation and glycosylation. The system gives very high recombinant protein yields and we often recommend it for proteins that are difficult to express in E. coli.
- Phase I: Gene Synthesis, Subcloning, and Baculovirus Paration
- Customer provides cDNA or optimize with any desired coding sequence and subclones it into a baculovirus transfer vector. Once cloned, all constructs are restriction digest and sequence verified. Transfer vectors are given to us and are used
to generate recombinant bacmids and P1, P2, and P3 virus stocks. CTSR needs 10ug of sterile DNA that has been cloned into Baculovirus expression system. We recommend using Life Technologies BAC TO BAC system.
- Phase II: Protein Expression and Purification
- We infect insect cells with high titer P3 viral stock and determine optimal conditions for protein expression and purification.
- Phase III: Larger Scale Protein Expression and Purification
- Should additional quantities of protein be required, we scale up expression by utilizing 2-10L Wave /bag system.
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