Expression, purification and crystallization of Dpr, a ferritin-like protein from the Gram-positive meningitis-associated bacterium Streptococcus suis.

Ferritin-like proteins form a novel family of bacterial proteins with diverse functions, such as DNA binding, iron storage and cell activation. A common structural feature of these proteins is their ability to form spherical dodecamers. Dpr is a ferritin-like protein from the Gram-positive bacterium Streptococcus suis. Full-length and truncated Dpr were expressed and purified as 6xHis-tag fusion proteins. Crystals of truncated Dpr suitable for X-ray diffraction analysis were obtained after the removal of the N-terminal affinity tag by thrombin cleavage. A complete data set to 2.3 A resolution was collected using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 104.3, b = 137.6, c = 142.1 A and 12 molecules in the asymmetric unit.

H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl.

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.