Validation of isoform-specific antibodies against PtAUREOs

For screening of PtAUREO knockdown or knockout cell lines highly specific antibodies are required which show very little cross-reactivity with the other isoforms or other unrelated proteins of P. tricornutum. An antiserum against PtAUREO1a had already been raised and had been successfully used for Western Blots of RNAi-induced PtAUREO1a knockdown strains (Schellenberger Costa et al., 2013b). Antisera against the other three isoforms were raised as well (Agrisera, Vännas, Sweden) and needed to be validated. The amino acid sequences of the four isoforms, the location of the conserved domains as well as the peptide chosen for immunization is depicted in Figure S2-1. Previous studies on PtAUREO1a used either French press or sonication to lyse cells. However, the use of the French press is not suitable for processing many samples, e.g. derived from different cultivation conditions and/or screening of transformed cell lines, due to time-consuming sample processing and cleaning procedure in between samples. Sonication also does not allow processing of multiple samples at once, and preparations varied widely in quality (data not shown). As an alternative, lysis with 2% SDS for 30 min at room temperature, which was successfully used for extraction of the cryptochrome CPF1 (Coesel et al., 2009), was also found not to be reliable for use with Aureochromes. Finally, the use of a bead mill and a strong lysis buffer containing urea, thiourea and SDS was found to be the preferred method, as there was very little variation between sample preparations and allowed lysis of multiple samples in parallel in a short time. Additionally, the use of chaotropic agents is known to destabilize DNA protein interactions, thus increasing solubility which facilitates extraction of Aureochromes. Using this protocol, each antibody produced a single band at the expected size (1a: 41.5 kDa, 1b: 46 kDa, 1c: 36 kDa, 2: 48 kDa) with seemingly little cross-reactivity among the different isoforms (see Figure 2-3).

Figure 2-3: Validation of the isoform-specific antisera using crude protein extract of P. tricornutum. Each antisera detects a single protein corresponding to the expected molecular weight (PtAUREO1a: 41.5 kDa;

PtAUREO1b: 46 kDa; PtAUREO1c: 36 kDa; PtAUREO2: 48 kDa)

2 Circadian rhythm of PtAureo transcripts and evaluation of isoform-specific antisera 17 To test whether the antibodies can also be used to detect their target protein in a native state Immunoprecipitation was tried under non-denaturing conditions and the elute analyzed by SDS-PAGE followed by silver staining (see Figure 2-4). A lower and upper band corresponding to the light and heavy chains of the antibody used for precipitation, as well as a third band presumably corresponding to the respective Aureochrome is visible. However, when analyzed by mass spectrometry, no Aureochrome could be identified, presumably due to a too low amount. Thus, the antibodies seem to have a strongly reduced affinity to the protein in its native state, at least under the conditions assayed.

Figure 2-4: Immunoprecipitation of PtAUREO1a/1b/2 using the antibodies raised against the respective isoforms, followed by silver staining. The upper and lower band correspond to the heavy and light chain of the antibody, whereas the middle band could correspond to the respective Aureochrome. Analysis by mass spectrometry did not yield Aureochromes, presumably due to a too low amount of protein.

To confirm that the detected bands correspond to the protein of interest and are not the result of an unintended cross reaction, E. coli overexpression constructs were generated for all four isoforms. As FMN binding of isoform PtAUREO2 was hypothesized to be restored by a single point mutation (Banerjee et al., 2016a), an additional overexpression construct, PtAUREO2_M301V, was generated by site-directed mutagenesis. Cell pellets as well as clarified cell lysates of PtAUREO1a-, PtAUREO1b- and PtAUREO1c-overexpressing cell lines were colored yellow instead of the standard brownish color, indicating high amounts of Flavin cofactors in the cell and therefore FMN-bound Aureochromes (see Figure 2-5). Wild type PtAUREO2-overexpressing cells on the other hand did not show this phenotype after induction, as expected, whereas overexpression of PtAUREO2_M301V lead to yellow coloring, confirming the hypothesis raised by Banerjee et al 2016. Thus, a single base pair change turned this photoreceptor into a non-light regulated protein. Whether it is regulated in

2 Circadian rhythm of PtAureo transcripts and evaluation of isoform-specific antisera 18 a different manner or performs a constitutive function should thus be studied in more detail in further studies.

Figure 2-5: Cell pellets or crude extracts of E. coli strains overexpressing PtAUREOs. Expectation for FMN binding capability of the different isoforms is indicated. A yellow color can be seen in protein extracts of PtAUREO1a/1b/1c as well as cells overexpressing PtAUREO2 M301V, but not wild type PtAUREO2.

Analysis of E. coli protein extracts prior to and after induction of PtAUREO expression with IPTG via SDS-PAGE followed by Coomassie staining (see Figure 2-6) showed appearance of bands not present prior to induction. Thus, expression of the proteins was confirmed. Western Blots using the crude protein extracts and all four antibodies showed a strong band when using the corresponding protein extract and very little cross-reactivity for the other isoforms, making them a valuable tool for the characterization of PtAUREO expression levels and screening of knockdown/knockout strains.

Figure 2-6: Validation of the isoform-specific antisera using E. coli strains expressing the different PtAUREO isoforms. A strong band (marked by an arrow) appears in each lane after induction of PtAUREO expression with IPTG (left). Each antisera labels the respective isoform with very little crossreactivity (right).

2 Circadian rhythm of PtAureo transcripts and evaluation of isoform-specific antisera 19