Cloning, expression and preliminary crystallographic studies of the potential drug target purine nucleoside phosphorylase from Schistosoma mansoni.
The parasite Schistosoma mansoni, unlike its mammalian hosts, lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. The gene encoding one enzyme of this pathway, purine nucleoside phosphorylase from S. mansoni (SmPNP) was identified, fully sequenced and cloned into the bacterial expression vector pMAL c2G to produce a protein in fusion with maltose-binding protein. The recombinant fusion protein was expressed at high levels and was purified in a single step by amylose resin affinity chromatography. After factor Xa cleavage, SmPNP was purified using a cation-exchange column and crystallized by hanging-drop vapour diffusion using polyethylene glycol 1500 as precipitant in the presence of 20% glycerol in acetate buffer. The use of the non-detergent sulfobetaine 195 (NDSB 195) as an additive had a marked effect on the size of the resulting crystals. Two data sets were obtained, one from a crystal grown in the absence of NDSB 195 and one from a crystal grown in its presence. The crystals are isomorphous and belong to the space group P2(1)2(1)2(1). It is intended to use the structures in the discovery and development of specific inhibitors of SmPNP.
Pereira
Humberto M
HM
Instituto de Física de São Carlos, Universidade de São Paulo, Brazil.
Cleasby
Anne
A
Pena S
Sérgio D J
SD
Franco G
Glória R
GR
Garratt
Richard C
RC
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011683:Purine-Nucleoside Phosphorylase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D012555:Schistosomiasis mansoni / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D011683:Purine-Nucleoside Phosphorylase