Supplementary MaterialsSI_Sequences

Supplementary MaterialsSI_Sequences. et al., 2008). The most readily useful tags can be used to deliver diverse chemical reporters with optimal properties, such as spectrally-distinct colors, high quantum yield and NSI-189 extinction coefficient (brightness), and photostability (Dempsey et al., 2001; Li and Vaughan, 2018). Tags that can bind to a variety of bright fluorophore ligands include the SNAP tag (Keppler et al., 2003; Gautier et al., 2008), Halo tag (Los et al., 2008), TMP tag (Miler et al., 2005), and FAPs (Szent-Gyorgyi et al., 2008). However, these protein tags are large NSI-189 (18C33 kDa), which can change protein folding, trafficking, and function (Brock et al., 1999; Costantini and Snapp, 2013; Huang et al., 2014; Johnson et al., 2015). A few peptide tags have been described for cell imaging, as exemplified by the tetracysteine tag, but these can have nonspecific interactions and limited color choices (Griffin et al., 1998; Gaietta et al., 2002; Cohen et al., 2011; Liu et al., 2014). An alternative approach is to use a peptide tag that forms a heterodimeric coiled-coil with a reporter peptide. This is the approach that we (Zane et al., 2017; Doh et al., 2018) as well as others (Tsutsumi et al., 2009; Nomura et al., 2010; Tsutsumi et al., 2011; Reinhardt et al., 2014 and 2015; Lotze et al., 2018) have used to label cellular proteins (Lotze et al., 2016; Yano and Matsuzaki, 2019). One advantage of this approach is that the genetic tag is usually small-just 4 to 7 kDa. A second advantage is usually that reporter peptide labeling is typically restricted to the cell surface, which is useful for labeling and tracking transmembrane receptors (Yano et al., 2008) in pulse-chase experiments (Doh et al., 2018; Lotze et al., 2018)). We named our coiled-coil tags Versatile Interacting Peptide (VIP) tags. Our first tag, VIP Y/Z, enabled the selective fluorescent labeling of target proteins in cell lysates and on live cells (Zane et al., 2017). Next we explained VIPER, which is usually comprised of a CoilE tag and a CoilR probe peptide. We showed that this probe peptide can be customized to the imaging application by conjugation to one of a number of reactive fluorophores and small molecules biotin). VIPER labeled sub-cellular structures in fixed cells and transmembrane receptors on live cells. Proteins could be imaged by FM or GAS1 correlative light and EM (CLEM) (Doh et al., 2018). For any genetic tag, it is important to place the tag at a location in the amino acid sequence where it will not interfere with the binding interactions, localization, folding, or function of the protein of interest. It is beyond the scope of this paper to dictate the location of the genetic tag for all those feasible protein targets. We recommend reading Erik Snapps paper Design and Use of Fluorescent Fusion Proteins NSI-189 in Cell Biology for any discussion on choosing a tag insertion site (Snapp, 2005). For VIP tags, we offer the following suggestions and recommendations for tag placement. For any new fusion protein, we recommend analyzing the localization, trafficking, and function to ensure that the tagged protein retains the same behavior as the untagged protein. Avoid placing the VIP tag in critical locations. In other words, tags should not be placed at catalytic residues, binding interfaces, or sites of post-translational modifications glycosylation, phosphorylation, zymogen cleavage, Furthermore, for secreted proteins, the tag should be positioned after the indication peptide in order to avoid getting cleaved. Cautious evaluation from the protein crystal framework, when available,.