Production and Purification of Recombinant Proteins from Escherichia Coli, Mammalian, Insect and Yeast Cells
Recombinant proteins are most commonly expressed in the following platforms:
- bacterial cells, commonly Escherichia coli
- Yeast (e.g., Pichia pastoris and Saccharomyces cerevisiae)
- Baculovirus expression vector systems (Autographa californica multiple nuclear polyhedrosis virus and insect cell hosts Spodoptera frugiperda or Trichoplusia ni)
- Mammalian cell systems (including a variety of transformed cell lines such as CHO and HEK293)
A bacterial system is the most commonly used expression system for production of recombinant proteins including antibodies. A transgenic mammalian system is used for extracellular expression of large amounts of recombinant proteins, reformatting scFv-FC and IgGs, and when the product contains post-translational modifications.
The AbET core provides the above services and advice on the following commonly occurring questions:
- Should the protein(s) be expressed in bacteria, yeast, insect cells or mammalian cells?
- Which expression vector should be used?
- If bacterial expression is used, which strain(s) should be chosen?
- Should one express the full-length protein or a fragment?
- Should the protein be tagged, and if so which affinity tag is the best?
- What is a good purification strategy, and what are the common pitfalls?
Mammalian proteins require the development of robust protein expression systems particularly for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds, distinct glycosylation patterns, other post translational modifications and folding requirements, expression in prokaryotic expression hosts can be problematic. In those cases, a mammalian cell expression systems is used.
AbET core provides advice on the following questions which are unique to mammalian expression systems.
- What kind of signal sequences is needed for protein secretion?
- Utilization of fusion tags for enhancing protein stability and purification and what kind of tag is most appropriate for the protein?
- What kinds of media formulation and optimization are required for high expression of protein?
- Which cell lines are optimal for the large-scale production?