Asparaginyl-tRNA synthetase from Thermus thermophilus HB8. Sequence of the gene and crystallization of the enzyme expressed in Escherichia coli.
Asparaginyl-tRNA synthetase from Thermus thermophilus HB8. Sequence of the gene and crystallization of the enzyme expressed in Escherichia coli.
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来自嗜热栖热菌 HB8 的天冬酰胺酰-tRNA 合成酶。
DOI：
10.1111/j.1432-1033.1996.0501u.x
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
R. Leberman
中科院分区：
文献类型：
--
作者：
L. Seignovert;M. Härtlein;R. Leberman
从极端嗜热栖热菌Thermus thermophilus HB 8中获得的天冬酰胺酰-tRNA合成酶基因（IIb类酶）已被克隆并测序。序列分析表明，该基因的开放阅读框编码438个氨基酸残基（50875 Da）。天冬酰胺酰-tRNA合成酶基因（asnS）中的密码子使用与栖热菌属细菌的蛋白质基因中的特征使用相似，并且密码子的第三位中的G+C含量高达94%。对T.嗜热菌的天冬酰胺酰-tRNA合成酶序列与其它细菌天冬酰胺酰-tRNA合成酶序列显示出高度相似性（30-55%同一性）。通过T.将嗜热菌asnS基因转化到大肠杆菌中，通过热处理和两步层析步骤过量产生并纯化至均一的耐热酶。所获得的蛋白质具有显著的热稳定性，并且在90 ° C下孵育1小时或在85 ° C下孵育21小时后保留其初始tRNA氨酰化活性的50%。通过气相扩散技术从聚乙二醇6000溶液中获得酶的晶体。这种晶体能吸收2.8埃以上的X射线。
The gene for the asparaginyl-tRNA synthetase, a class IIb enzyme, from the extreme thermophile Thermus thermophilus HB8 has been cloned and sequenced. Sequence analysis revealed an open reading frame that codes for a protein of 438 amino acid residues (50875 Da). Codon usage in the asparaginyl-tRNA synthetase gene (asnS) is similar to the characteristic usage in the genes for proteins from bacteria of the genus Thermus, and the G+C content in the third position of the codons is as high as 94%. The amino acid sequence of asparaginyl-tRNA synthetase from T. thermophilus shows high similarity with other bacterial asparaginyl-tRNA synthetase sequences (30-55% identity). By expression of the T. thermophilus asnS gene in Escherichia coli, the thermostable enzyme was overproduced and purified to homogeneity by heat treatment and two chromatography steps. The protein obtained is remarkably thermostable and retains 50% of its initial tRNA aminoacylation activity after 1 h of incubation at 90 degrees C or 21 h at 85 degrees C. Crystals of the enzyme were obtained from polyethylene glycol 6000 solutions by vapour diffusion techniques. The crystals diffract X-rays beyond 2.8 A.