Dynamism of cellulose synthases

GhCesA2是编码4-β-葡萄糖基转移酶的细菌纤维素合成酶基因的同源基因。GhCesA2的中心催化区在巴斯德毕赤酵母中以可溶性蛋白的形式表达。重组蛋白的相对分子质量为10 0 kDa，经内切糖苷酶H处理后，分子量降至85 kDa。在棉花下胚轴提取液存在下，重组GhCesA2能催化葡萄糖从二磷酸葡萄糖转移到未知产物，但产物不是β-1，4-葡聚糖。棉花(Gossypium hirsutom L.)利用杆状病毒系统在昆虫细胞中表达了纤维素合成酶基因GhCesA2，并测定了该基因产物在天然状态下的流体力学性质。重组蛋白可被1%的Triton X-100从感染的Sf9细胞膜部分溶解。凝胶过滤层析和H_2O/D_2O蔗糖梯度沉淀…的结果More n表明，重组GhCesA2具有两种大小。我们计算了蛋白质-洗涤剂复合物的部分比体积、斯托克斯半径和相对分子质量，并估计了两种蛋白质的相对分子质量分别为142,000和285,000。结果表明，重组GhCesA2形成了一种同源二聚体，由于其重组蛋白的纯化和结晶程序尚未建立，因此纤维素合酶的三维结构仍很困难。在本研究中，我们成功地利用杆状病毒表达系统在SF9细胞中表达了组氨酸标记的BCSA(细菌纤维素合成酶的A亚单位)。通过SDS-PAGE和Western blotting对重组蛋白的表达进行了鉴定和验证，发现用His标签纯化重组蛋白有望实现规模化生产。我们现在正在进入下一阶段，以更精确地测量蛋白质，例如，通过电子显微镜和X射线小角散射来测量蛋白质的活性以及尺寸分布。较少

GhCesA2 is a cotton (Gossypium hirsutum) homolog of bacterial cellulose synthase gene that encodes cellulose 4-β-glucosyltransferase. The central catalytic region of GhCesA2 was expressed as a soluble protein in methylotrophic yeast Pichia pastoris. The molecular size of the recombinant protein was 100 kDa, which decreased to 85 kDa after the treatment with endoglycosidase H. The recombinant GhCesA2 catalyzed transfer of glucose from UDP-glucose into unknown products in the presence of the extract of cotton hypocotyls, but the products were not β-1,4-glucan. The putative cotton (Gossypium Hirsutum L.) cellulose synthase gene GhCesA2was expressed in insect cells using baculovirus systems and the hydrodynamic properties of the gene product were determined in its native state. The recombinant protein could be solubilized with 1% Triton X-100 from the membrane fraction of infected Sf9 cells. The results obtained from gel filtration chromatography and H_2O/D_2O sucrose gradient sedimentatio … More n showed that the recombinant GhCesA2 had two types in size. We calculated the partial specific volume, Stokes radius and molecular weight of the protein-detergent complex and estimated the molecular weight of the two proteins as 142,000 and 285,000. The result suggested that the recombinant GhCesA2 form a homo-dimer.Three-dimensional structure of cellulose synthase remains difficult because none of purification and crystallization procedures for its recombinant protein has not been established. In this study, we succeeded in expressing a Histidine-tagged BcsA.(subunit A of a bacterial cellulose synthase) using a bacurovirus expression system in SF9 cells. The expression of this recombinant protein was assessed and verified by SDS-PAGE and western blotting, and found promissing for mass production by purifying them with His tags. We are now stepping in the next stage to measure more precisely the proteins, for instance, the activity as well as the size distribution either by electron microscopy and X-ray small angle scattering. Less