Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon.
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.
Wakagi
T
T
Department of Biotechnology, University of Tokyo, Japan. atwakag@hongo.ecc.u-tokyo.ac.jp
Oshima
T
T
Imamura
H
H
Matsuzawa
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003062:Codon / Q000737:chemistry; D016384:Consensus Sequence; D019641:DNA, Archaeal / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019848:Gene Expression Regulation, Archaeal; D043564:Inorganic Pyrophosphatase; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012347:RNA, Transfer, Arg / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics