Immobilized Tryptophanyl-tRNA Synthetase from Bacillus subtilis.
In order to investigate the recognition mechanism and the relationship between structure and function of tRNA(Trp) with tryptophanyl-tRNA synthetase (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 subjected to 3 successive rounds of immobilized TrpRS column selection with SELEX method, resulting in a sharp increase of the percentage of the RNA pool that could bind immobilized TrpRS from 4.3% for the first round pool to 14.7% for the third-round pool. After sequencing the third-round RNA pool, a RNA secondary structure resembling the structure of the acceptor stem in tRNA(Trp) was obtained though the selection. All the results indicated that immobilized TrpRS could be used as an affinity chromatography matrix and was qualified for the SELEX of a RNA pool simulating tRNA(Trp) molecule.
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. yxjin@sunm.shcnc.ac.cn
Feng
F
Xu
Li
L
Chen
Quan-Sheng
QS
Liu
You-Xin
YX
Jin
De-Bao
DB
Wang