We have successfully used HEK293 suspension cells for many years, for both secretory and intracellular protein expression. We routinely use the baculovirus/insect cell system, working with the flashBACTM technology from Oxford Expression Technologies. Both of these, and in particular HEK293 cells, enable the most authentic post-translational modifications for human proteins which is key for any protein expression service. Secreted proteins are purified from cell culture media. Our systems use protein free media, greatly simplifying the purification process.
We also frequently use E. coli because of the speed and quantity of protein that can be achieved when a particular protein expresses well. We manipulate the standard T7 based expression vector systems to maximise soluble expression. We can refold proteins from E. coli derived inclusion bodies.
All the standard affinity chromatographic procedures are used regularly with a variety of different tags. The design and selection of the most appropriate affinity tag can have a significant impact on the purification process and protein yields achieved. In addition, proteins are purified according to size, charge, hydrophobicity and other properties depending on the protein characteristics. A suite of Akta systems are available for the work.
As part of our protein production service, proteins are analysed by a variety of methods. SDS-PAGE is used under both reducing and non-reducing conditions as required, to look for disulphide bonds. Purity and size in solution is determined using size exclusion chromatography. Protein concentration is established using absorbance at 280nm, correcting for the extinction coefficient. Liquid chromatography mass spectrometry is used in two ways. If the protein will “fly” in the mass spectrometer –the intact mass of the protein is determined to search for post-translational modifications. The peptide map can be determined after proteolytic digestion. The thermal stability of the protein will also be monitored if needed.