Overexpression and Periplasmic Transport of 5-Enolpyruvylshikimate 3-Phosphate Synthase in E. coli

대장균에서 5-Enolpyruvylshikimate 3-Phosphate Synthase의 대량 발현 및 Periplasmic Space로의 Transport

  • 김남일 (충북대학교 자연과학대학 생명과학부) ;
  • 임재윤 (충북대학교 자연과학대학 생명과학부) ;
  • 조태주 (충북대학교 자연과학대학 생명과학부)
  • Published : 1997.03.01

Abstract

5-Enolpyruvylshikimate 3-phosphate(EPSP) synthase is the sixth enzyme of the shikimate pathway that synthesizes aromatic amino acids. The enzyme is a primary target for the glyphos'lte which is a broad-spectrum and environmetally safe herbicide. As a first step toward development of glyphpsate-resistant EPSP synthase, the EPSP synthase gene(aroA) was amplified by polymerase chain reaction and cluned into pET-25b vector. In this construct. designated pET-aro, the aroA gene is expressed under control of strong T7 promoter. and the EPSP synthase is produced as a fusion protein with pelB leader at N-terminus and HSV-tag and His-tag at C-terminus. When the pET-aro clone was induced to produce the enzyme, it was found that the EPSP synthase was successfully exported to peri plasmic space. The periplasmic transport was greatly dependent on the induction temperatures. Among the induction temperatures examined($25^{\circ}C$, $30^{\circ}C$, $34^{\circ}C$ and $37^{\circ}C$). induction at $34^{\circ}C$ gave rise to maximal periplasmic transport. The recomhinant EPSP synthase could have been purified hy $Ni^{2+}$ -affinity chromatography using the His-tag. and detected hy anti-HSV -tag antibody. The recombinant EPSP synthase also hound to phosphocellulose resin and was eluted hy shikimate 3-phosphate and phosphoenolpyruvate. as expected. The recombinant EPSP synthase purified from phosphocellulose resin showed typical EPSP synthase activity.

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