Test & optimisation of expression
A quick test of expression undertaken at 48 hours post-infection will provide a reasonable indication of whether a protein is expressed to reasonable levels or not, however, by varying a number of parameters it is possible to optimise the levels of expression for a particular protein. It is difficult to estimate in advance whether a protein will be expressed to high levels. At OET we routinely provide optimisation of expression by varying five parameters:
Baculovirus vector – can compare flashBAC™,flashBACGOLD™ and flashBACULTRA™ to see if the gene deletions present in GOLD and ULTRA enhance yield and/or quality. The decision to compares virus vector needs to be taken at the start of the project.
Baculovirus promoter – Although the polyhedrin andp10 promoters are inherently the strongest we can use, some more difficult to express proteins give better yields using an earlier promoter that are active when the cell is less compromised from virus infection. We can offer to compare the very late polyhedrin with the late p6.9. This decision needs to be made at the start of the project as it will affect which transfer vectors are used.
Cell line – Proteins yield and quality can vary considerably between cell lines and we offer three cell lines: Sf21, Sf9, SuperSf9 and Tni. This decision can be made after the results of the quick analysis of protein expression are known.
Multiplicity of infection – Protein yield and quality can also vary with multiplicity of infection, and sometimes a lower MOI can yield higher quality protein. We normally compare MOIs of 0.5, 2 and 5. This decision can be made after the results of the quick analysis of expression are known.
Time to harvest – Small shake cultures are set up in the appropriate cell line(s). Samples for analysis of protein synthesis are taken at 3 or 4 times points e.g. 0, 24, 48, 72 and 96 hours after infection. A control (mock-infected) sample at 48 hpi is always included. Pellets and/or culture medium can be analysed by Western blots using anti-tag antiserum or primary antiserum provided by the client (or purchased by OET on behalf of the client).
At the end of the optimisation stage, OET staff will discuss the results with the client and agree upon the optimal conditions for protein production.