枯草杆菌TrpRS的固定化及亲和RNA筛选

为研究 t RNATrp与色氨酰 - t RNA合成酶 ( Trp RS)的相互识别及其结构与功能的关系 ,纯化了枯草杆菌 Trp RS,并用溴化氰活化的 Sepharose4B将 Trp RS固定化 ,固定化 Trp RS的蛋白回收率为 95.5% ,活力回收率为 31 .3% .研究了固定化 Trp RS的酶学性质 ,其热稳定性和贮存稳定性方面均比液相 Trp RS有了较大的提高 ,最适温度、最适 p H均有一定程度的增大 ,工作稳定性良好 .以固定化 Trp RS为亲和层析介质 ,对含有 2 0个核苷酸随机序列 ,长度为 56个核苷酸的单链RNA随机库进行了三轮筛选 .实验结果表明 ,固定化 Trp RS可以作为 SELEX亲和层析介质 ,进行模拟 t RNATrp分子的 RNA随机库的 SELEX筛选 .

Immobilization of TrpRS from Bacillus subtilis and Selection of RNA Aptamers

In order to investigate the recognition mechanism and the relationship between structure and function of tRNA Trp with TrpRS,TrpRS from Bacillus Subtilis was purified and immobilized on CNBr\|activated Sepharose 4B.Protein recovery and activity recovery of the immobilization were 95 5% and 31 3%,respectively.Properties of immobilized TrpRS were studied in detail.The thermal stability and the shelf stability of immobilized TrpRS were much higher than those of the native TrpRS.Besides these,the immobilized TrpRS,with good operation stability,had increased optimum temperature and optimum pH.A 56\|base single\|stranded RNA library containing 20 consecutive completely randomized bases was subject to 3 successive rounds of immobilized TrpRS column selection with SELEX method.All the results indicated that immobilized TrpRS could be used as an affinity chromatography matrix and was qualified for the SELEX of an RNA pool simulating tRNA Trp molecule.