The Core is currently using four complementary expression systems: bacteria, Baculovirus, Schneider cells and CHO cells. The choice for either one of these expression systems is guided by prior experience with similar proteins, literature searches and some specific scientific concerns (e.g. glycosylation of extracellular proteins). The overall general rules for expression system choice are as follows:
Bacterial expression: cytoplasmic and nuclear proteins.
Baculovirus expression: cytoplasmic proteins not expressed in bacteria, extracellular domains of surface receptors, nuclear proteins.
Schneider's cell expression (referred to as "Fly Expression System or FES": extracellular domains of surface receptors, multimeric proteins.
CHO expression: proteins requiring mammalian glycosylation for function, proteins not expressed in insect systems or bacteria.
Most recombinant proteins are produced with a histidine tag to allow expression screening and purification. Consequently, the first step of purification is most often NI-NTA-agarose chromatography. Alternative approaches include GST and Fc fusions among others. Further purification is carried out by low-pressure chromatography using combinations of ion exchange, gel permeation, and/or hydrophobic interaction techniques.
Identity confirmation of recombinant proteins is carried out by trypsin digestion/mass spectrometry analysis.
Mice, rats and hamsters are used to satisfy species barriers.
Two adjuvants are alternatively used: Titermax (Sigma) or α-galactosylceramide.
Animals are immunized three times at two week intervals
and boosted four days before fusion. Fusions are carried out following a standard protocol using P3 myeloma cells as
the fusion partner.
The initial screen is carried out by ELISA using the recombinant protein as a target. Coating, washing and reading of ELISA plates is done automatically using an Evolution EP3 liquid handling system. Positive clones are expanded to 24 well plates and re-tested by ELISA before further characterization, which includes Western blot, immunoprecipitation, and FACS analysis whenever amenable. Selected clones are frozen and stored in liquid nitrogen. Clones to be distributed to the general public are subcloned by limiting dilution and re-tested before freezing.
Polyclonal antibodies are sometimes produced. Rabbits are used for this purpose. Immunoglobulins are purified on target antigen columns.