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. 2020 Jul 21;295(37):13065–13078. doi: 10.1074/jbc.RA120.014266

Figure 3.

Figure 3.

Production and purification of recombinant CntB and CntA proteins, l-carnitine depletion assay, TMA determination, and NADH depletion assay. A, SDS-PAGE analysis of CntB His tag affinity purification using Co2+-loaded chelating Sepharose. Lane 1, E. coli cells after IPTG induction; lane 2, supernatant after ultracentrifugation; lane 3, elution fraction of WT CntB. Affinity purification of GST-tagged CntA was also done: lane 4, E. coli cells after IPTG induction; lane 5, supernatant after ultracentrifugation; lanes 6–15, purified CntA WT and variants C86A, H88A, C106A, H109A, E205Q, E205D, H208A, H213A, and D323A after affinity chromatography on GSH-Sepharose (untagged target proteins eluted after on-column PreScission protease cleavage). Lanes M, molecular mass marker; relative molecular masses (×1,000) are indicated. B, l-carnitine depletion assay. A standard assay containing 5 μm purified CntA, 20 μm purified CntB, and 300 μm l-carnitine at 37 °C was initiated by the addition of 2 mm the electron donor NADH (black trace) or NADPH (blue trace). Samples were prepared at least in triplicates and collected after 15, 30, 60, and 90 s, and l-carnitine concentrations were determined colorimetrically in a coupled carnitine acetyltransferase assay (see Experimental procedures). Control reactions in the absence of CntA (red trace), electron donor (green trace), or O2 (orange trace; proteins purified under strict anaerobic conditions) were performed. C, TMA analysis by GC. Top, a standard assay in the presence of 2 mm l-carnitine and 5 mm NADH was incubated at 37 °C for 2 min. The reaction was stopped in the presence of 1 M perchloric acid and subjected to GC analyses. Control reactions in the absence of CntB (middle) or NADH (bottom) were performed. D, NADH depletion assay in the presence of 2 μm purified CntA and CntB, 300 μm l-carnitine, and 200 μm NADH (black trace). The absorption at 340 nm was continuously monitored (black trace). Control reactions in the absence of O2 (orange trace), CntB (purple trace), and CntA (red trace) were performed.