Yoon Bo-Young, Jiao Li, Moon Hyung Ryong, Cha Jaeho, Ha Nam-Chul
College of Pharmacy and Research Institute for Drug Development, Pusan National University, Busan 609-735, Republic of Korea.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Jan 1;68(Pt 1):56-8. doi: 10.1107/S1744309111047099. Epub 2011 Dec 24.
The β-N-acetylglucosaminidase CbsA was cloned from the thermophilic Gram-negative bacterium Thermotoga neapolitana. Although CbsA contains a family 3 glycoside hydrolase-type (GH3-type) catalytic domain, it can be distinguished from other GH3-type β-N-acetylglucosaminidases by its high activity towards chitobiose. The homodimeric CbsA contains a unique domain at the C-terminus for which the three-dimensional structure is not yet known. In this study, CbsA was overexpressed and the recombinant protein was purified using Ni-NTA affinity and gel-filtration chromatography. The purified CbsA protein was crystallized using the vapour-diffusion method. A diffraction data set was collected to a resolution of 2.0 Å at 100 K. The crystal belonged to space group R32. To obtain initial phases, the crystallization of selenomethionyl-substituted protein and the production of heavy-atom derivative crystals are in progress.
β-N-乙酰氨基葡萄糖苷酶CbsA是从嗜热革兰氏阴性细菌那不勒斯嗜热栖热菌中克隆得到的。尽管CbsA含有一个3型糖苷水解酶家族（GH3型）催化结构域，但它对壳二糖具有高活性，这使其有别于其他GH3型β-N-乙酰氨基葡萄糖苷酶。同型二聚体CbsA在C末端含有一个独特的结构域，其三维结构尚不清楚。在本研究中，CbsA被过量表达，重组蛋白通过镍-亚氨基三乙酸（Ni-NTA）亲和层析和凝胶过滤层析进行纯化。纯化后的CbsA蛋白采用气相扩散法进行结晶。在100 K下收集了分辨率为2.0 Å的衍射数据集。该晶体属于R32空间群。为了获得初始相位，硒代甲硫氨酸取代蛋白的结晶和重原子衍生物晶体的制备正在进行中。
