메타게놈에서 발굴한 프로모터를 장착한 새로운 항시발현 벡터의 가치평가
Evaluation of Novel Constitutive Expression Vectors Equipped with Mined Promoters from Metagenome.
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The choice of expression vector is very important for industrial production of proteins. Therefore, the systematic mining of promoters over a wider range of genetic resource and/or host is required. We previously reported a novel bidirectional reporting system(pBGR) for the isolation of promoters from metagenome and screened useful promoters that functioned constitutively in E. coli under general culture conditions. Among them, three promoter sequences including each upstream region were amplified by PCR and used to construct new expression vectors. To facilitate subcloning, a multi-cloning site was incorporated into the downstream region of the revere primer sequence. At these sites, GFP, esterase and β-glucosidase were subcloned and analyzed the constitutive expression ability of new promoter in terms of protein solubility and expression level. As a result, these vectors expressed the proteins constitutively to a level of 2~3% of the total cell protein in soluble fraction (>80 %). This study suggested that excavation of metagenomic promoters for construction of expression vector in a certain strain could provide a way for the development of the expression systems.
Abstract
The choice of expression vector is very important for industrial production of proteins. Therefore, the systematic mining of promoters over a wider range of genetic resource and/or host is required. We previously reported a novel bidirectional reporting system(pBGR) for the isolation of promoters from metagenome and screened useful promoters that functioned constitutively in E. coli under general culture conditions. Among them, three promoter sequences including each upstream region were amplified by PCR and used to construct new expression vectors. To facilitate subcloning, a multi-cloning site was incorporated into the downstream region of the revere primer sequence. At these sites, GFP, esterase and β-glucosidase were subcloned and analyzed the constitutive expression ability of new promoter in terms of protein solubility and expression level. As a result, these vectors expressed the proteins constitutively to a level of 2~3% of the total cell protein in soluble fraction (>80 %). This study suggested that excavation of metagenomic promoters for construction of expression vector in a certain strain could provide a way for the development of the expression systems.
참고문헌 (24)
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