To generate a fluorescently-tagged and biotinylatable human γTuRC, the coding region for full-length human GCP2 (amino acids 1-902) was amplified by PCR using its cDNA as template (NM_001256617.1, Origene). The mTagBFP (blue fluorescent protein, Evrogen) coding sequence was also amplified by PCR. Both PCR-amplified sequences were cloned into a pLVX-Puro vector (Clonetech) using Gibson assembly (In-Fusion cloning, Takara), to form GCP2_G5A_TEV_G5A_mTagBFP_G5A_BAP, an expression construct for GCP2 which is C-terminally tagged with mTagBFP and biotin acceptor peptide (BAP: GLNDIFEAQKIEWHE), both separated from GCP2 by a TEV protease cleavage site. Glycine linkers (G5A) were placed between sequences. To facilitate the in vivo biotinylation of tagged γTuRC E. coli biotin ligase BirA was cloned into a pLVX-IRES-Hyg vector (Clonetech) using Gibson assembly to form HA_ G5A_BirA; an expression construct of BirA with an HA-tag added to the BirA N-terminus, separated by a G5A-linker. Primers used for cloning are listed in the Key Resources Table.
Consolati T., Locke J., Roostalu J., Chen Z.A., Gannon J., Asthana J., Lim W.M., Martino F., Cvetkovic M.A., Rappsilber J., Costa A, & Surrey T. (2020). Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure. Developmental Cell, 53(5), 603-617.e8.
+ Open protocol