Wakagi T, Oshima T, Imamura H, Matsuzawa H
Department of Biotechnology, University of Tokyo, Japan.
Biosci Biotechnol Biochem. 1998 Dec;62(12):2408-14. doi: 10.1271/bbb.62.2408.
The gene encoding an extremely stable inorganic pyrophosphatase from Sulfolobus sp. strain 7, a thermoacidophilic archaeon, was cloned and sequenced. An open reading frame consisted of 516 base pairs coding for a protein of 172-amino acid residues. The deduced sequence was supported by partial amino acid sequence analyses. All the catalytically important residues were conserved. A unique 17-base-pair sequence motif was found to be repeated four times in frame in the gene, encoding a cluster of acidic amino acids essential for the function. Although the codon usage of the gene was quite different from that of Escherichia coli, the gene was effectively expressed in E. coli. Coexpression of tRNA(Arg), cognate for the rare codon AGA in E. coli, however, further improved the production of the enzyme, which occupied more than 85% of the soluble proteins obtained after removal of heat denatured E. coli proteins.
对嗜热嗜酸古菌硫化叶菌属菌株7中编码一种极其稳定的无机焦磷酸酶的基因进行了克隆和测序。一个开放阅读框由516个碱基对组成，编码一个含有172个氨基酸残基的蛋白质。推导的序列得到了部分氨基酸序列分析的支持。所有催化重要残基均保守。发现一个独特的17碱基对序列基序在该基因中按读框重复四次，编码对该功能至关重要的一簇酸性氨基酸。尽管该基因的密码子使用与大肠杆菌有很大不同，但该基因在大肠杆菌中能有效表达。然而，共表达大肠杆菌中与稀有密码子AGA对应的tRNA(Arg)，进一步提高了该酶的产量，该酶在去除热变性的大肠杆菌蛋白后获得的可溶性蛋白中占比超过85%。
