35s met cys expre35s35s protein labelling mix

In vitro transcription of Rattus norvegicus OTC precursor was performed using pSP65/pre-OTC71 (link). To generate a plasmid for in vitro transcription of OTC-Δ, pSP65/pre-OTC was digested with Bgl II25 (link), releasing a 255 bp insert and the linear plasmid ligated to generate pSP65/pre-OTC-Δ. To generate radiolabelled pre-OTC and pre-OTC-∆, in vitro transcription and translation was performed. Briefly, pSP65/pre-OTC and pSP65/pre-OTC-Δ were digested with Sma I then used as template to synthesise mRNA using SP6 RNA polymerase (Promega). The ³⁵S-labelled precursor proteins were synthesised from pre-OTC and pre-OTC-∆ mRNA in rabbit reticulocyte lysate (Promega), supplemented with 11 μCi [³⁵S]Met/Cys EXPRE³⁵S³⁵S protein labelling mix (specific activity >1000 Ci/mmol; Perkin Elmer).

HeLa (ATCC® CCL2™) cells were grown and maintained at 70–90% confluency at 37 °C with 5% (v/v) CO₂ in Dulbecco’s Modified Eagle Medium (DMEM; ThermoFisher Scientific) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) for a maximum of one month. Mitochondria were isolated from HeLa cells as described^{24 (link)}. In vitro import reactions were performed essentially as described^{25 (link)}. Wild type and mutant precursor CLPP (preCLPP) proteins were radiolabelled in the presence of 11 µCi [³⁵S]Met/Cys EXPRE³⁵S ³⁵S Protein Labelling Mix (specific activity >1000 Ci/mmol) (Perkin Elmer, Waltham, MA, USA), using the TnT® SP6 Coupled Reticulocyte Lysate System (Promega, Australia), according to the manufacturer’s instructions. Following import, the proteins were separated by SDS-PAGE (see below), gels were then dried, and the radiolabelled proteins visualised by digital autoradiography using a Typhoon Trio Molecular Imager (GE Healthcare).