Figure 2. Analyses on antibody expression and assembly in P. tricornutum.

A. Twelve independent P. tricornutum transfectants were screened for expression of heavy and light chain of the antibody. Western Blot analyses demonstrate that expression levels are very similar. For further analyses clone number 12 was selected as expression seems to be slightly enhanced. B. Western Blot analyses demonstrate that both antibody chains are expressed in P. tricornutum cells and assemble into complete antibodies. Under reduced conditions both antibody chains are detected separately, whereas in non-reduced SDS-PAGE a high molecular weight signal is detected, which corresponds to the fully-assembled antibody. C. Monoclonal antibodies are expressed under a NO₃ ⁻-inducible promoter and different induction periods were tested to get highest expression levels. Best induction period turned out to be 1–2 days (lane 2+3), which was interestingly similar to expression levels with constant induction from the very beginning (lane 7). D. Antibodies expressed in P. tricornutum were purified using protein A-Sepharose beads. 5% of the eluate was loaded on a SDS-PAGE demonstrating that purification is very efficient with no contaminations detected by Coomassie or Silver Staining. Western Blot analyses with subsequent incubation with the lectin ConA revealed that both antibody chains get glycosylated in the ER of P. tricornutum. ConA – Concanavalin A, d – days.