Pereira Humberto M, Cleasby Anne, Pena S Sérgio D J, Franco G Glória R, Garratt Richard C
Instituto de Física de São Carlos, Universidade de São Paulo, Brazil.
Acta Crystallogr D Biol Crystallogr. 2003 Jun;59(Pt 6):1096-9. doi: 10.1107/s090744490300773x. Epub 2003 May 23.
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.
曼氏血吸虫这种寄生虫与其哺乳动物宿主不同，缺乏嘌呤生物合成的从头合成途径，其嘌呤需求依赖于补救途径。编码该途径中一种酶的基因，即曼氏血吸虫嘌呤核苷磷酸化酶（SmPNP）被鉴定、完全测序并克隆到细菌表达载体pMAL c2G中，以产生与麦芽糖结合蛋白融合的蛋白。重组融合蛋白大量表达，并通过直链淀粉树脂亲和层析一步纯化。经Xa因子切割后，使用阳离子交换柱纯化SmPNP，并在乙酸盐缓冲液中20%甘油存在的情况下，以聚乙二醇1500作为沉淀剂，通过悬滴气相扩散法使其结晶。使用非离子型磺基甜菜碱195（NDSB 195）作为添加剂对所得晶体的大小有显著影响。获得了两组数据集，一组来自在无NDSB 195情况下生长的晶体，另一组来自在有NDSB 195情况下生长的晶体。这些晶体是同晶型的，属于空间群P2(1)2(1)2(1)。旨在利用这些结构来发现和开发SmPNP的特异性抑制剂。
