Rapid Biotin Labeling with TurboID-CpOGA Constructs

HEK293T cells (Meisen CTCC) were cultured in a DMEM/high glucose medium (Biological Industries, 01-052-1A) with 10% FBS (VISTECH, SE100-B) at 37℃ under 5% CO₂. The CpOGA^CD and CpOGA^DM sequences were codon optimized to Homo sapiens and Drosophila using Jcat (Grote et al., 2005 (link)). The fragments of TurboID-CpOGA^CD and TurboID-CpOGA^DM (TurboID-CpOGA^CD/DM) were PCR amplified and cloned into pCDH-CMV-HA vectors, respectively. For lentivirus preparation, HEK293T cells were transfected with TurboID-CpOGA^CD/DM plasmid with the packaging plasmids pPAX2 and pMD.2G using Polyethylenimine Linear (PEI, Polysciences, 24765). The PEI-containing medium was replaced with fresh serum-containing DMEM medium after 8 hr, and the viral supernatants were collected 48 hr and 72 hr post-transfection. The viral supernatants were centrifuged at 10,000 g for 1 hr at 4℃, and the pellet was dissolved in PBS (Biological Industries, 02-023-1A). HEK293T cells were infected in six-well plates and selected with 1 µg/mL Puromycin (Selleck, s7417) in the medium for at least 5 days. For biotin labeling, the TurboID-CpOGA^CD or TurboID-CpOGA^DM expressing HEK293T cells were labeled with 10–100 µM biotin (Merck, B4501) in the medium for 15 min to 3 hr. Labeling was stopped by placing cells on ice and washing cells three times with PBS (Biological Industries, 02-023-1A).