Tnt quick coupled transcription and translation system

The TnT Quick Coupled Transcription and Translation system (Promega, Madison, USA) was used to generate [³⁵S]-labeled HBsAg protein from the S gene cloned in pcDNA3.1 vector, using the T7 promoter present on the vector.20 (link) Briefly, rabbit reticulocyte lysate was mixed with 1 mg of plasmid DNA, 2 µL of [³⁵S]-methionine (Amersham, 1,000 Ci/mmol at 10 mCi/mL) and nuclease-free water to a final volume of 50 mL. The reaction mixture was incubated for 90 min at 30 ℃. Expression of each construct was determined by 12 % SDS-PAGE. After fixation for 30 min, the gel was treated with Amplify solution (Amersham, Little Chalfont, UK) and the radio-labeled protein was detected by exposure to X-ray film.

In vitro transcription and translation were carried out using TNT Quick coupled Transcription and Translation system (Promega, Amedison, WI, USA). For in vitro translation, pcDNA-HA-PIG3, pcDNA-Myc-p53 and pcDNA3-MDM2 expression vector were used. One microgram of each construct was used per reaction of the TNT-Quick coupled reticulocyte lysate system (Promega), following the manufacturer’s instructions. The synthesized proteins were used in vitro ubiquitination and in vitro protein binding assay.