Figure 1 Complete set of PBPs identified with Boc-FL in whole cells of L. monocytogenes. Samples of whole cells (100 μg of Selonsertib molecular weight total protein) were labeled with Boc-FL at concentrations of 0 (1), 0.5
(2), 1 (3), 2.5 (4), 5 (5), 10 (6), 50 μM (7) and 50 μM plus 100 μg/ml ampicillin (8). Labeled bands were detected directly on the gel, quantified, and their molecular mass estimated. The affinity of each band for Boc-FL (ID50) was estimated from their fluorescence as a function of the concentration of Boc-FL. The name of the PBP corresponding to each band is indicated on the right, while the positions of molecular weight markers (bars) and unspecific bands (arrowheads) are shown on the left. Table 2 Competition binding assay and affinity of different PBPs of L. monocytogene s for Boc-FL PBP Boc-FL Kd50 a Ampicillin c PPBA1 (PBP1) >10 μM 95 PBPB2 (PBP2) 0.25 μM 90 PBPB1 (PBP3) 0.25 μM 0 PBPA2 (PBP4) 0.25 μM 90 PBPB3 >20 μM 95 PBPD1 (PBP5) 5.0 μM 0 PBPC1 >20 μM 100 PBPC2 >20 μM 100 PBPD3 n.a. n.a. PBPD2 2.5 μM b 0 b a affinity of the respective bands for Boc-FL LCZ696 estimated from their fluorescence as a function of the concentration of Boc-FL (Kd50) b obtained with purified recombinant Lmo2812 c percentage of Boc-FL binding capacity remaining after sample was preincubated with 100 μg/ml ampicillin Characterization of protein Lmo2812 (PBPD2) Gene lmo2812 was amplified by PCR
from the wild-type EGD strain and cloned in vector pET30a without
its putative lipobox signal peptide. Expression of the His-tagged fusion protein in E. coli BL21(DE3) cells was induced with IPTG and it was purified from cell lysates on a nickel affinity column. The recombinant Lmo2812 protein was eluted from the column by GDC-0941 mouse washes with 250 and 500 mM imidazole. These two fractions were combined and further purified on a desalting Branched chain aminotransferase column, yielding 4 mg/ml of pure protein. The purified protein was incubated with different concentrations of Boc-FL (0.25, 0.5, 2.5, 5 and 10 μM). Saturation binding studies showed that Lmo2812 covalently bound Boc-FL, indicating that the recombinant protein retained its authentic activity. Lmo2812 was the major band on gels, with a slower migrating minor band thought to represent a dimeric form (Figure 2). Figure 2 Purified recombinant L. monocytogenes Lmo2812 (PBPD2) identified with Boc-FL. Samples of purified recombinant Lmo2812 (10 μg) were labeled with Boc-FL at concentrations of 0 (1), 0.25 (2), 0.5 (3), 2.5 (4), 5 (5) and 10 μM (6). Labeled bands were detected directly on the gel, quantified and their molecular mass estimated. The affinity of the bands for Boc-FL (Kd50) was estimated from their fluorescence as a function of the concentration of Boc-FL. The names of the bands are indicated on the right, and the positions of the molecular weight markers are shown on the left.