Immobilized
Tryptophanyl-tRNA Synthetase from Bacillus subtilis
XU Feng, CHEN Li, LIU Quan-Sheng, JIN You-Xin*, WANG De-Bao
( State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry,
the Chinese Academy of Sciences, Shanghai 200031, China )
Abstract In order
to investigate the recognition mechanism and the relationship between structure
and function of tRNATrp 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 tRNATrp 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 tRNATrp
molecule.
Key words immobilized enzyme； TrpRS； SELEX
Received: July 19,
1999 Accepted: October 13, 1999
The project was supported by the Natural Science Foundation of China (No.
39730120) and the Chinese Academy of Sciences(No. KJ 951-B1-610)
* Corresponding author: Tel, 86-21-64374430; Fax, 86-21-64338357; e-mail, [email protected]