The use of M115 and M135 as alternative translation initiation sites was supported by the finding that no HBP35 translational product was

detected in the hbp35 [M115A and M135A] insertion mutant (KDP170). Moreover, recombinant HBP35 proteins with a C-terminal histidine-tag were produced in an E. coli strain expressing the hbp35 gene and purified by a histidine-tag purification system. Immunoblot analysis revealed that the purified products contained 40-, 29-, and 27-kDa proteins immunoreactive to the anti-HBP35 anitibody. Edman sequencing revealed that the N-terminal amino acid residue of the recombinant 27-kDa protein was M135 (Additional file 4). SAHA solubility dmso Hemin binding site of rHBP35 proteins Shibata et al. [7] found that a purified rHBP35 protein (Q22-P344) could bind hemin and

that HBP35 was suggested to possess a putative heme binding sequence (Y50CPGGK55). To determine the hemin binding region of HBP35, we constructed and purified rHBP35 (Q22-P344), rHBP35 (Q22-P344 with C48S and C51S) and truncated rHBP35 (M135-P344) proteins with N-terminal histidine-tags using a histidine-tag purification system and carried out hemin binding assays using a hemoprotein peroxidase assay. As shown in QNZ solubility dmso Figure 4B, all of the rHBP35 (Q22-P344), rHBP35 (Q22-P344 with C48S and C51S) and truncated rHBP35 (M135-P344) proteins were found to have hemin binding ability, implying that the hemin binding site is located in check details M135-P344 of HBP35 protein. Figure 4 Hemin binding of various rHBP35 proteins. Two μg each of rHBP35(Q22-P344) (lane 1), rHBP35 (Q22-P344 with C48S C51S) (lane 2), truncated rHBP35(M135-P344) (lane 3), or lactoferrin as a negative control (lane 4) was treated with or without 1.5 μl of 1.25 mM hemin for 2 h at room temperature. A, CBB staining; B, peroxidase activity staining. Arrowheads indicate the hemin binding proteins. Effect of hemin depletion on growth of the hbp35 mutant Since

HBP35 protein is a hemin-binding protein, we determined the contribution of HBP35 proteins to acquisition or intracellular storage Silibinin of heme. The hbp35 insertion mutant, the full length deletion mutant, the complemented strain which was constructed by replacing the intact hbp35 gene into the hbp35 full length deletion mutant, and the wild-type strain were hemin-starved after being grown in enriched BHI broth containing hemin (Figure 5). Hemin starvation resulted in retardation of the growth of the hbp35 mutants compared to that of the wild type, whereas the complemented strain partially recovered the growth retardation of the hbp35 deletion mutant under the hemin-depleted condition. Even under the hemin replete condition, the hbp35 mutants grew more slowly than the wild type, suggesting that HBP35 plays a role in hemin utilization in a sufficient hemin concentration (5 μg/ml). Figure 5 Growth in hemin-containing BHI broth (0-48 h) and hemin-free BHI broth (after 48 h).