Ultraflex 3 maldi

Vector pET28a and the amplicon of qnrVC5 (from V. fluvialis BD146) each were separately double-digested with BamHI and XhoI. Subsequent to digestion, the dephosphorylated vector and qnrVC5 insert were ligated, the ligation mixture was electroporated in E. coli JM109 cells and the transformed cells were plated on LB plates containing kanamycin (50 μg ml^-1) to obtain the recombinants. Expression of QnrVC5 protein was studied by induction of E. coli BL21 (λDE3) cells harboring the recombinant clones with 1mM IPTG for 2 h at 37°C, followed by SDS-PAGE analysis of total cell lysates. The protein band corresponding to QnrVC5 was excised from the gel and subjected to trypsin digestion. The authenticity of this protein was then confirmed by peptide mass fingerprinting on Bruker Ultraflex III MALDI instrument. The protein identification was done through Mascot software.

Purified recombinant PgMntR was resolved by SDS-PAGE and subjected to in-gel tryptic digestion as described previously [23 (link)]. Peptide mass fingerprinting analysis was carried out on an Ultraflex III MALDI (matrix-assisted laser dissociation ionization) TOF/TOF (time of flight) mass spectrometer (Bruker Daltonics) and proteins were identified using the Mascot v 2.2 search engine (Matrix Science, London, UK) [24 (link)].