Excerpts

The new “mox” (monomeric oxidation protected) PSFP, moxEos3.2, was resistant to inappropriate disulphide bond formation when expressed in the ER, while the parent “wt” mEos3.2 formed a ladder of covalent oligomers, with noticeably very little protein in the monomeric state. We also confirmed that the alanine mutations did not alter the monomericity of mEos3.2 by performing a CytERM assay for FP oligomerization. Despite these successful outcomes, we noticed that moxEos3.2 was visually much dimmer in the green and red states in comparably transfected and expressing cells. Furthermore, this mox form bleached substantially faster than the wt parent in both the green and red states. Taken together, the optimization of mEos3.2 for the secretory pathway had impaired the performance of the protein.