Ghosh S, Ghosh R, Das P, Chattopadhyay D
Department of Biochemistry, Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, India.
Protein Expr Purif. 2001 Feb;21(1):40-8. doi: 10.1006/prep.2000.1319.
Giardia lamblia, the ancient eukaryote does not have nucleolus but produces the fibrillarin protein that may be used for pre-rRNA processing. The nucleoli of eukaryotes contain complex population of small nucleolar RNAs, known as snoRNAs, several of which are required for rRNA processing. This report describes the full-length cloning of fibrillarin gene from Giardia lamblia, using RTPCR and the production of recombinant fibrillarin protein in Escherichia coli strain BL21 (DE3) as N-terminal His-tag protein. The condition for production of soluble protein was standardized. The expressed protein was purified by using Ni-chelation chromatography and used for functional studies. The small nuclear RNAs (snRNAs), RNA D, RNA J, and RNA H, containing box C, box D, and box C/D, respectively, of Giardia were also cloned by RTPCR. Antibody raised against the recombinant protein was used to identify the fibrillarin in giardial nuclear extract. The interaction of snRNAs with recombinant fibrillarin was followed using North-Western hybridization. Gel electrophoresis mobility shift assay demonstrated that bacterially expressed protein may participate in the in vitro interaction with RNA J, RNA H, and RNA D. Our results indicate that the recombinant fibrillarin by itself is able to bind and does not require the involvement of any other protein for this binding to the three snRNAs.
蓝氏贾第鞭毛虫，这种古老的真核生物没有核仁，但能产生可能用于前体核糖体RNA加工的纤维蛋白。真核生物的核仁包含复杂的小核仁RNA群体，即snoRNAs，其中几种是核糖体RNA加工所必需的。本报告描述了利用逆转录聚合酶链反应从蓝氏贾第鞭毛虫中全长克隆纤维蛋白基因，并在大肠杆菌BL21（DE3）菌株中生产作为N端组氨酸标签蛋白的重组纤维蛋白。对可溶性蛋白的生产条件进行了标准化。表达的蛋白通过镍螯合层析进行纯化，并用于功能研究。蓝氏贾第鞭毛虫分别含有C框、D框和C/D框的小核RNA（snRNAs），即RNA D、RNA J和RNA H，也通过逆转录聚合酶链反应进行了克隆。针对重组蛋白产生的抗体用于鉴定贾第虫核提取物中的纤维蛋白。利用蛋白质印迹杂交追踪snRNAs与重组纤维蛋白的相互作用。凝胶电泳迁移率变动分析表明，细菌表达的蛋白可能参与与RNA J、RNA H和RNA D的体外相互作用。我们的结果表明，重组纤维蛋白自身能够结合，并且在与这三种snRNAs结合时不需要任何其他蛋白的参与。
