RSM was employed to optimize the screened induction conditions that enhanced the recombinant bromelain production.
Therefore, it is important to screen different induction times, especially if other means of increasing productivity should be evaluated.
Even so, the advantage of initial screening based solely on DsRed fluorescence is that second-stage screening following induction of the linked transgene is limited to a handful of promising clones, rather than tens or hundreds [ 37].
Some screening of induction potential can be conducted by the use of human hepatocytes, but supply and variable response limits their use.
First, we performed four primary screens: LOPAC inhibition and induction screens and Kinase Inhibitors Library inhibition and induction screens.
Knockdown of ATG5 reduced CPT-induced AVO formation, thus validating AVO as a reliable screen for autophagy induction.
Another compound validated in our induction screen, previously used in studies with RBC, is sorafenib tosylate (Bay 43 9006).
For the induction screen of the Kinase Inhibitor Library we selected hits for further validation based on z-score, which accounted for 21 compounds in total.
Therefore, it is important to screen at different induction times for phosphate-limited and -unlimited cultures to evaluate if a phosphate limitation is generally suitable to enhance specific productivity.
Most compounds were considered validated hits when their median of the replicates (Rep Median score) was smaller than 1, in case of inhibition screens and higher than 1 in case of induction screens and the adjusted P-value (Padj score) was smaller than 0.1 with the exception of eight compounds which were validated according to cell scatter.
We performed inhibition and induction screens in parallel since we aimed at identifying molecules that induce RBC vesiculation (induction screens) and molecules that inhibit vesiculation induced by ionomycin (inhibition screens).
