Krishnan G, Morabito M A, Moczydlowski E
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066, USA.
Toxicon. 2001 Feb-Mar;39(2-3):291-301. doi: 10.1016/s0041-0101(00)00127-6.
Saxiphilin is a plasma protein from the bullfrog (Rana catesbiana) that binds saxitoxin (STX), a causative agent of paralytic shellfish poisoning. Saxiphilin is homologous to transferrin and consists of two internally homologous domains called the N-lobe and the C-lobe. STX binds to a single site in the C-lobe of saxiphilin. In this study, cloned genes coding for recombinant saxiphilin and C-lobe saxiphilin were modified to contain two tandemly located affinity tags, Flag epitope (DYKDDDDK) and His(6) (HHHHHH), at the protein C-terminus and were expressed in cultured insect cells using baculovirus vectors. Both tagged proteins are readily detected on immunoblots by anti-Flag monoclonal antibody. Flag-His(6)-tagged saxiphilin was purified to homogeneity using Ni(2+)-chelate affinity chromatography and Heparin Sepharose chromatography. Equilibrium analysis of [3H]STX binding to tagged saxiphilin and tagged C-lobe saxiphilin gave K(D) values of 0.75 and 2.7 nM, respectively. Flag-His(6)-tagged saxiphilin was also utilized in a microtiter well solid-phase assay with Reacti-bind metal chelate plates to measure [3H]STX binding and binding competition by unlabeled STX. Such Flag-His(6)-tagged derivatives of saxiphilin have many possible applications in the assay of STX and related toxinological research.
石房蛤毒素结合蛋白是一种来自牛蛙（美国牛蛙）的血浆蛋白，它能结合石房蛤毒素（STX），后者是麻痹性贝类中毒的病原体。石房蛤毒素结合蛋白与转铁蛋白同源，由两个内部同源结构域组成，分别称为N叶和C叶。STX结合到石房蛤毒素结合蛋白C叶的一个单一部位。在本研究中，编码重组石房蛤毒素结合蛋白和C叶石房蛤毒素结合蛋白的克隆基因经修饰后，在蛋白质C末端含有两个串联的亲和标签，Flag表位（DYKDDDDK）和His(6)（HHHHHH），并使用杆状病毒载体在培养的昆虫细胞中表达。两种带标签的蛋白都能通过抗Flag单克隆抗体在免疫印迹上轻松检测到。带有Flag-His(6)标签的石房蛤毒素结合蛋白通过Ni(2+)-螯合亲和色谱和肝素琼脂糖色谱纯化至同质。[3H]STX与带标签的石房蛤毒素结合蛋白和带标签的C叶石房蛤毒素结合蛋白的平衡分析得出的K(D)值分别为0.75和2.7 nM。带有Flag-His(6)标签的石房蛤毒素结合蛋白还用于使用Reacti-bind金属螯合板的微量滴定孔固相测定中，以测量[3H]STX结合以及未标记的STX的结合竞争。这种带有Flag-His(6)标签的石房蛤毒素结合蛋白衍生物在STX测定和相关毒理学研究中有许多可能的应用。
