Figure 1.

A modularized expression system enables purification of 2xFLAG-tagged recombinant γ-TuRC. (a) Schematic representation of the 14-spoke vertebrate γ-TuRC. Colours: GCP2 (light blue), GCP3 (dark blue), γ-tubulin (orange/yellow), GCP4 (brown), GCP5 (green), GCP6 (purple), actin (red) and luminal bridge (pink). (b) Cloning strategy for the recombinant γ-TuRC using the MultiBac vectors pACEBac1, PIDC, pIDK and pIDS with polyhedrin (polH) expression cassette. Human genes of the γ-TuRC were inserted via Infusion cloning. The ‘modules', plasmids with one or two genes of interest, were combined via subsequent Cre-recombination. Construct 1 for γ-TuRC expression consists of 2xFLAG-GCP5, GCP6, GCP4, TUBG1 and ACTB. This construct was used for bacmid and virus production and for protein expression. It was complemented with construct 2 coding for MZT1, GCP2 and GCP3. (c) Recombinant γ-TuRC was isolated via FLAG affinity purification and FLAG peptide elution in a single-step protocol and used for subsequent characterization using negative stain EM as well as biochemical approaches. Scheme of the γ-TuRC indicating the approximate position of the 2xFLAG tag at the N-terminus of GCP5. Colours as in (a). (d) Section of a negative stain EM micrograph of human recombinant γ-TuRC after FLAG affinity purification. Yellow boxes indicate exemplary γ-TuRC particles. Scale bar: 100 nm. (e) Section of Coomassie Blue-stained SDS-PAGE gel of γ-TuRC elution after FLAG affinity purification. (f) Cell lysate and FLAG affinity-purified recombinant γ-TuRC (FLAG elution) were probed using immunoblotting against the indicated antibodies. See electronic supplementary material, figure S2 for uncropped images.