Test & Optimisation Of Expression

A quick test of expression undertaken at 48 hours of expression 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 VLPs. It is difficult to estimate in advance whether a protein will be expressed to high levels. At NanoExpo we routinely provide optimisation of expression by varying five parameters:

Baculovirus vector vs mammalian vector and bacterial vector – a comparison of the level of production in different hosts. The decision to compares virus vector needs to be taken at the start of the project.

Cell line (insect vs mammalian vs bacteria) – Proteins yield and quality can vary considerably between cell lines and we offer three insect cell lines: Sf21, Sf9, SuperSf9 and Tni, mammalian cell lines: HEK293T, CHO, EpiCHO, HEKfreestyle, bacteria: BL21, BL21STAR, ARCTIC, GAMMI, ROSETAGAmMI. This decision can be made after the results of the quick analysis of protein expression.

Multiplicity of infection in baculovirus system – 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 NanoExpo on behalf of the client).

At the end of the optimisation stage, NanoExpo staff will discuss the results with the client and agree upon the optimal conditions for protein production.