人CD96第一个IgV结构域的表达、结晶及晶体学分析

Expression, crystallization and crystallographic study of the 1st IgV domain of human CD96

摘要:

CD96 (Tactile) 是一个主要表达在活化的T细胞、NK细胞上的免疫受体分子。它属于免疫球蛋白超家族，胞外区有3个免疫球蛋白结构域 (V1，V2/C和C)。最近的研究表明，CD96的第一个IgV结构域 (CD96V1) 在细胞粘附和NK细胞介导的杀伤过程中起着重要的作用。文中构建了人类CD96第一个IgV样结构域 (hCD96V1) 的表达载体，并将它在大肠杆菌BL21 (DE3) 菌株中以包涵体形式表达。通过包涵体体外复性，得到了CD96可溶性蛋白。分析超速离心结果证明CD96可溶蛋白在体外以二聚体形式存在。采用座滴气相扩散法获得了衍射质量较好的CD96蛋白晶体，衍射分辨率为1.9?，晶体属于空间群P21，晶胞参数为a=35.1，B=69.5，C=49.6?，α=γ=90°，β=105.4°。CD96晶体及衍射数据的获得为后续的结构和功能研究奠定了基础。

Abstract:

CD96 (Tactile) is an adhesion receptor expressed mainly on activated T cells, NK cells. As a family member of the immunoglobulin-like cell receptor, CD96 consists of three immunoglobulin-like domains (V1, V2/C and C) in the extracellular region. Recent studies have shown that the 1st IgV domain of CD96 (CD96V1) plays an essential role in cell adhesion and NK cell-mediated killing. In this study, the 1st IgV domain of human CD96 (hCD96V1) was cloned and expressed in Escherichia coli (BL21). The soluble protein was obtained by refolding of the hCD96V1 inclusion bodies. From analytical ultracentrifugation, we could predict that CD96 V1 maily exists as dimer with approximate molecular weight of 26.9?kDa. The protein was then successfully crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.9?? resolution and belonged to space group P21, with unit-cell parameters a=35.1, b=69.5, c= 49.6?, α=γ=90°, β=105.4°.