Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase

Abstract

Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. The inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 A and the Ser/Thr phosphatase crystals to 2.65 A. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail.

Figure 1

Expression of the serine/threonine phosphatase (STP) fusion protein, the inorganic pyrophosphatase (PPase) fusion protein and a pGEX4T3 control followed as a function of time. Samples were taken at 1.5, 2.5 and 5.5 h post-induction. Lane 1, molecular-weight markers (kDa) (Fermentas SMO431). Lanes 2, 5 and 8, expression of the empty vector control (pGEX4T3; C). Lanes 3, 6 and 9, expression of the SaSTP fusion protein (S). Lanes 4, 7 and 10, expression of the SaPPase fusion protein (P).