Recombinant Expression and Biotinylation of EcoKMcrB-N

A SalI-SacI fragment containing coding sequence for EcoKMcrB-N [18 (link)] was synthesized (GeneWiz) and cloned into the pET52 Expression Vector (Millipore) and transformed into the T7 Express cell line (NEB). The expressed recombinant protein has an N-teminal Strep tag and a C-terminal His tag from the pET52 vector to facilitate purification. Cultures were propagated at 37°C until OD₆₀₀ is 0.4–0.6 and induced with IPTG at a final concentration of 0.05 mM. Induction was performed at 30°C on shaker for 4 hours and the cells were harvested by centrifugation. Lysates were prepared by Lysozyme treatment on ice and freeze-thaw. Lysates were clarified by centrifugation for 30 minutes followed by purification with Strep-Tactin Superflow Plus (Qiagen).
The tagged McrB-N was biotin labeled with the EZ-Link Sulfo-NHS-biotin kit (Pierce, Rockford, IL) following the manufacturer’s instructions. The extent of biotinylation was evaluated using the HABA assay (Pierce). Each mole of the tagged McrB-N was found to contain 6 mole of biotin.