Rathore D, Nayak S K, Batra J K
Immunochemistry Laboratory, National Institute of Immunology, New Delhi, India.
FEBS Lett. 1996 Sep 2;392(3):259-62. doi: 10.1016/0014-5793(96)00825-3.
Restrictocin is a toxin produced by the fungus Aspergillus restritus. The DNA coding for restrictocin was isolated from the host by polymerase chain reaction and cloned into a T7 promoter-based expression vector. The protein was overproduced in Escherichia coli and remained insoluble in the cell in the form of inclusion bodies. Recombinant restrictocin was purified in large amounts, by a simple denaturation-renaturation protocol involving a redox system, with typical yields of 45 mg/l of original culture. Restrictocin could be secreted into the bacterial medium using ompA, pelB and LTB signal sequences. Among the three signal sequences, ompA was found to be the most efficient in secreting the recombinant protein. The protein secreted into the extracellular medium was properly processed as evident by the amino-terminal sequencing. Recombinant restrictocin was readily purified to homogeneity from either the medium or inclusion bodies by simple chromatographic techniques and was found to be functionally as active as the native fungal protein in inhibiting the eukaryotic translation.
限制酶是由局限曲霉产生的一种毒素。通过聚合酶链反应从宿主中分离出编码限制酶的DNA，并将其克隆到基于T7启动子的表达载体中。该蛋白在大肠杆菌中过量表达，并以包涵体的形式在细胞内保持不溶性。通过一种涉及氧化还原系统的简单变性-复性方案大量纯化重组限制酶，原始培养物的典型产量为45mg/L。使用ompA、pelB和LTB信号序列，限制酶可以分泌到细菌培养基中。在这三个信号序列中，发现ompA在分泌重组蛋白方面最有效。通过氨基末端测序可以明显看出，分泌到细胞外培养基中的蛋白得到了正确加工。通过简单的色谱技术，重组限制酶很容易从培养基或包涵体中纯化至同质，并且发现在抑制真核翻译方面其功能与天然真菌蛋白一样活跃。
