Otrelo-Cardoso Ana Rita, Nair Rashmi R, Correia Márcia A S, Rivas Maria G, Santos-Silva Teresa
Rede de Química e Tecnologia/Centro de Química Fina e Biotecnologia (REQUIMTE/CQFB), Department of Chemistry, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica 2829-516, Portugal.
Int J Mol Sci. 2014 Jul 2;15(7):11783-98. doi: 10.3390/ijms150711783.
The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P21 space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.
TupABC系统参与细胞对钨的摄取，属于ABC（ATP结合盒）型转运系统。TupA组分是一种周质蛋白，可结合钨酸根阴离子，然后由TupB组分利用ATP水解作为能量来源（由ModC组分催化的反应）将其转运穿过膜。我们报道了阿拉斯加脱硫弧菌G20 TupA的异源表达、纯化、亲和结合常数的测定及结晶。tupA基因（基因座标签Dde_0234）被克隆到pET46肠激酶/不依赖连接的克隆（LIC）表达载体中，构建体用于转化BL21（DE3）细胞。优化了TupA的表达和纯化，最终每升培养基可获得10 mg可溶性纯蛋白。进行了非变性聚丙烯酰胺凝胶电泳，结果表明TupA能结合钨酸根和钼酸根离子，与硫酸根、磷酸根或高氯酸根无明显相互作用。通过等温滴定量热法对金属结合进行的定量分析与这些结果一致，此外还表明TupA对钨酸根的亲和力高于钼酸根。使用悬滴气相扩散法，该蛋白在30%（w/v）聚乙二醇3350存在下结晶。晶体的X射线衍射分辨率超过1.4 Å，属于P21空间群，晶胞参数a = 52.25 Å，b = 42.50 Å，c = 54.71 Å，β = 95.43°。已找到分子置换解，目前该结构正在优化中。
