Protein expression and functionality.
A) Western blots of HIV gag protein without [opt] and with [msg] message expressed in HEK293 cells. Equal amounts of protein from 5 independent transfections were analyzed from cell supernatants (top panels) and cell lysates (bottom panels), with actin detection serving as a control. B) Western blot of HEK293 lysates expressing GFP from optimized genes without [opt] and with [msg] embedded watermarks, including the appended human codon ranking sequence [msg+cut] (see Fig. 3), an encrypted watermark [msg enc] (see Table 2) and with a longer embedded message [msg long], also employing amino acids with 2 or 3 alternative codons (CDEFHIKNQY). Protein expression was quantified by densitometry. Results are derived from five independent experiments. C) Fluorescence microscopy images of GFP transfected into tobacco leaves show no visible differences in cellular location and only little variation in abundance. D) GST-T7 RNA polymerase [opt] and [msg] expressed in triplicates in E. coli was analyzed by SDS-PAGE and Coomassie staining (top panel) or Western blotting using a specific T7 RNA polymerase antibody (α-T7RNAP; lower panel). Equal amounts of purified T7 RNA polymerase were used for in vitro transcription. Synthesized RNA detected with a molecular beacon in real-time directly revealed almost identical RNA polymerase activities mediated by T7 [opt] (blue line) and T7 [msg] (orange line).