York J D, Chen Z W, Ponder J W, Chauhan A K, Mathews F S, Majerus P W
Division of Hematology-Oncology, Washington University School of Medicine, St Louis, MO 63110.
J Mol Biol. 1994 Feb 18;236(2):584-9. doi: 10.1006/jmbi.1994.1167.
Bovine inositol polyphosphate 1-phosphatase, a monomeric protein with a molecular mass of 44,000 Da, hydrolyzes the 1-position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. The low abundance of inositol polyphosphate 1-phosphatase in tissues has precluded structural studies requiring large quantities of enzyme. We used recombinant Baculovirus harboring the cDNA of bovine inositol polyphosphate 1-phosphatase to infect Spodoptera frugiperda (Sf9) insect cells. Recombinant protein (25 mg per 1 x 10(9) cells) was purified to homogeneity. The enzyme produced in Sf9 cells was similar to the native purified protein as determined by immunoblotting catalytic properties, and inhibition by lithium ions. Crystals of the purified recombinant enzyme were grown by vapor diffusion. Precession photography was used to determine the parameters of inositol polyphosphate 1-phosphatase crystals. The tetragonal crystals belong to the space group P4(1) or P4(3), have unit cell dimensions of a = b = 51.6 A, c = 143.3 A, alpha = beta = gamma = 90 degrees, and contain one molecule per asymmetric unit. We have collected a complete diffraction data set extending to 2.3 A and are currently attempting to solve the three-dimensional structure of bovine inositol polyphosphate 1-phosphatase using a multiple isomorphous replacement strategy.
牛肌醇多磷酸1-磷酸酶是一种分子量为44,000道尔顿的单体蛋白，可水解肌醇1,3,4-三磷酸和肌醇1,4-二磷酸1位上的磷酸基团。由于组织中肌醇多磷酸1-磷酸酶的丰度较低，使得需要大量酶的结构研究受到限制。我们利用携带牛肌醇多磷酸1-磷酸酶cDNA的重组杆状病毒感染草地贪夜蛾（Sf9）昆虫细胞。重组蛋白（每1×10⁹个细胞25毫克）被纯化至同质。通过免疫印迹、催化特性和锂离子抑制作用测定，Sf9细胞中产生的酶与天然纯化蛋白相似。通过气相扩散法培养纯化的重组酶晶体。利用进动摄影确定肌醇多磷酸1-磷酸酶晶体的参数。四方晶体属于空间群P4₁或P4₃，晶胞参数为a = b = 51.6 Å，c = 143.3 Å，α = β = γ = 90°，每个不对称单元包含一个分子。我们已经收集了分辨率达到2.3 Å的完整衍射数据集，目前正尝试使用多重同晶置换策略解析牛肌醇多磷酸1-磷酸酶的三维结构。
