Chen S, Court D L
Department of Biochemistry, Fourth Military Medical University, Xi'an, Shaanxi, China.
Chin J Biotechnol. 1992;8(2):82-91.
The reason for low content of RNaseIII in E. coli is that RNaseIII has a negative feedback action on its own synthesis by cutting off the transcripts of its own gene, rnc, at 5'-terminal. On this basis, the scheme for overproduction of RNaseIII was designed. The 5'-flanking sequence of rnc gene transcribing to form a secondary structure which could be degraded by RNaseIII was removed, and the whole coding sequence, including the translational initiation signal, was reserved and put under the control of lambda PL promoter. The constructed plasmid pCR21, which contain the recombined rnc gene, overproduced RNaseIII, which covered over 65% of the total cell protein and formed inclusive bodies in E. coli after induction at 42 degrees C. By using the characteristics of solubility of the protein, electrophoretic pure RNaseIII was obtained with a simple procedure, including lysis of the bacterial cells, washing precipitates of the lysate repeatedly at low temperature and low salt concentration, and dissolving and passing through Q-Sepharose FF column in high salt concentration at room temperature. The yield of purified RNaseIII was 10-12 mg per 100 ml culture, and lambda sib transcripts were cut at special sites. RNaseIII possessing the activity of binding ATP is reported.
大肠杆菌中核糖核酸酶III（RNaseIII）含量低的原因是，RNaseIII通过在其自身基因rnc的转录本5'端进行切割，对自身合成产生负反馈作用。在此基础上，设计了过量生产RNaseIII的方案。去除了rnc基因转录形成可被RNaseIII降解的二级结构的5'侧翼序列，保留了包括翻译起始信号在内的整个编码序列，并置于λPL启动子的控制之下。构建的含有重组rnc基因的质粒pCR21过量生产RNaseIII，在42℃诱导后，RNaseIII占大肠杆菌总细胞蛋白的65%以上，并形成包涵体。利用该蛋白的溶解性特点，通过简单的步骤获得了电泳纯的RNaseIII，包括裂解细菌细胞、在低温和低盐浓度下反复洗涤裂解物沉淀，以及在室温下高盐浓度下溶解并通过Q-Sepharose FF柱。纯化后的RNaseIII产量为每100 ml培养物10 - 12 mg，且λsib转录本在特定位点被切割。据报道，RNaseIII具有结合ATP的活性。
