放線菌ATPヌクレオチド3'-ピロホスホキナーゼ(PPKase)遺伝子の大腸菌における発現と宿主菌に与える影響

To elucidate the expression mechanism and to attempt application of Streptomyces ATP nucleotide 3'-pyrophosphokinase (PPKase) gene in heterologous hosts, we expressed PPKase gene in E. coli cells. This was carried out by inserting a DNA fragment including PPKase gene downstream of the lac promoter in a multi-copy plasmid pUC119. The resultant plasmid, pUP3 was introduced into E. coli JM109. When the cells were incubated in the presence of IPTG, PPKase protein was found to be expressed and secreted into the periplasmic space. PPKase promoter was not functional in E. coli like most Streptomyces promoters. The increased activity of PPKase in pUP317, a deletion plasmid from pUP3, indicated that 5'-flanking sequence was very important to express PPKase gene. Various 3'-pyrophosphoryl nucleotides were detected, and growth inhibition was observed in PPKase-producing E. coli cells. Further studies are necessary for practical application of PPKase and its gene.放線菌PPKase遺伝子を大腸菌で発現させ, 活性のあるPPKase蛋白を生成させることができた. 今回の実験結果ではPPKaseのプロモーターは大腸菌では認識されず, 放線菌のプロモーターは大腸菌では機能しないというこれまでの報告と一致していた. また, pUP317のPPKase発現に対しては5’-非翻訳領域の長さのみならず塩基配列そのものも大きな影響を与えることが示された. さらに, 菌体内にはPPKaseの反応生成物と思われる3'- ピロリン酸型ヌクレオチドが検出された. また, PPKaseの誘導発現に伴い宿主に生育抑制が起きることも明らかとなった. 今後, PPKaseの発現によって宿主菌内に起こる種々の変化について検討を進めていきたい

Expression of Streptomyces ATP Nucleotide 3\u27 -pyrophosphokinase (PPKase) Gene in E. coli and its Influence upon the Host Cells

To elucidate the expression mechanism and to attempt application of Streptomyces ATP nucleotide 3\u27-pyrophosphokinase (PPKase) gene in heterologous hosts, we expressed PPKase gene in E. coli cells. This was carried out by inserting a DNA fragment including PPKase gene downstream of the lac promoter in a multi-copy plasmid pUC119. The resultant plasmid, pUP3 was introduced into E. coli JM109. When the cells were incubated in the presence of IPTG, PPKase protein was found to be expressed and secreted into the periplasmic space. PPKase promoter was not functional in E. coli like most Streptomyces promoters. The increased activity of PPKase in pUP317, a deletion plasmid from pUP3, indicated that 5\u27-flanking sequence was very important to express PPKase gene. Various 3\u27-pyrophosphoryl nucleotides were detected, and growth inhibition was observed in PPKase-producing E. coli cells. Further studies are necessary for practical application of PPKase and its gene.放線菌PPKase遺伝子を大腸菌で発現させ, 活性のあるPPKase蛋白を生成させることができた. 今回の実験結果ではPPKaseのプロモーターは大腸菌では認識されず, 放線菌のプロモーターは大腸菌では機能しないというこれまでの報告と一致していた. また, pUP317のPPKase発現に対しては5’-非翻訳領域の長さのみならず塩基配列そのものも大きな影響を与えることが示された. さらに, 菌体内にはPPKaseの反応生成物と思われる3\u27- ピロリン酸型ヌクレオチドが検出された. また, PPKaseの誘導発現に伴い宿主に生育抑制が起きることも明らかとなった. 今後, PPKaseの発現によって宿主菌内に起こる種々の変化について検討を進めていきたい